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Sample records for aldosterone synthase expression

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

    SciTech Connect

    Okamoto, Mitsuhiro . E-mail: mokamoto@mr-mbio.med.osaka-u.ac.jp; Nonaka, Yasuki; Takemori, Hiroshi; Doi, Junko

    2005-12-09

    11{beta}-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 11{beta}-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.

  2. Progress towards clinically useful aldosterone synthase inhibitors.

    PubMed

    Cerny, Matthew A

    2013-01-01

    Owing to the high degree of similarity between aldosterone synthase (CYP11B2) and cortisol synthase (CYP11B1), the design of selective inhibitors of one or the other of these two enzymes was, at one time, thought to be impossible. Through development of novel enzyme screening assays and significant medicinal chemistry efforts, highly potent inhibitors of CYP11B2 have been identified with selectivities approaching 1000-fold between the two enzymes. Many of these molecules also possess selectivity against other steroidogenic cytochromes P450 (e.g. CYP17A1 and CYP19A1) as well as hepatic drug metabolizing P450s. Though not as well developed or explored, inhibitors of CYP11B1, with selectivities approaching 50-fold, have also been identified. The therapeutic benefits of affecting the renin-angiotensin-aldosterone system have been well established with the therapeutically useful angiotensin-converting enzymes inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists. Data regarding the additional benefits of an aldosterone synthase inhibitor (ASi) are beginning to emerge from animal models and human clinical trials. Despite great promise and much progress, additional challenges still exist in the path towards development of a therapeutically useful ASi.

  3. Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2)

    PubMed Central

    Schwab, Dietmar; Delporte, Marie-Laure; Palermo, Giuseppe; Amrein, Kurt; Mohr, Susanne; De Vera Mudry, Maria Cristina; Brown, Morris J.; Ferber, Philippe

    2017-01-01

    Primary hyperaldosteronism is a common cause of resistant hypertension. Aldosterone is produced in the adrenal by aldosterone synthase (AS, encoded by the gene CYP11B2). AS shares 93% homology to 11β-hydroxylase (encoded by the gene CYP11B1), responsible for cortisol production. This homology has hitherto impeded the development of a drug, which selectively suppresses aldosterone but not cortisol production, as a new treatment for primary hyperaldosteronism. We now report the development of RO6836191 as a potent (Ki 13 nmol/L) competitive inhibitor of AS, with in vitro selectivity >100-fold over 11β-hydroxylase. In cynomolgus monkeys challenged with synthetic adrenocorticotropic hormone, single doses of RO6836191 inhibited aldosterone synthesis without affecting the adrenocorticotropic hormone–induced rise in cortisol. In repeat-dose toxicity studies in monkeys, RO6836191 reproduced the adrenal changes of the AS−/− mouse: expansion of the zona glomerulosa; increased expression of AS (or disrupted green fluorescent protein gene in the AS−/− mouse); hypertrophy, proliferation, and apoptosis of zona glomerulosa cells. These changes in the monkey were partially reversible and partially preventable by electrolyte supplementation and treatment with an angiotensin-converting enzyme inhibitor. In healthy subjects, single doses of RO6836191, across a 360-fold dose range, reduced plasma and urine aldosterone levels with maximum suppression at a dose of 10 mg, but unchanged cortisol, on adrenocorticotropic hormone challenge, up to 360 mg, and increase in the precursors 11-deoxycorticosterone and 11-deoxycortisol only at or >90 mg. In conclusion, RO6836191 demonstrates that it is possible to suppress aldosterone production completely in humans without affecting cortisol production. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01995383. PMID:27872236

  4. Aldosterone does not modify gene expression in human endothelial cells.

    PubMed

    Verhovez, A; Williams, T A; Morello, F; Monticone, S; Brizzi, M F; Dentelli, P; Fallo, F; Fabris, B; Amenta, F; Gomez-Sanchez, C; Veglio, F; Mulatero, P

    2012-03-01

    The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

  5. A novel aldosterone synthase inhibitor ameliorates mortality in pressure-overload mice with heart failure.

    PubMed

    Furuzono, Shinji; Meguro, Masaki; Miyauchi, Satoru; Inoue, Shinichi; Homma, Tsuyoshi; Yamada, Keisuke; Tagawa, Yoh-Ichi; Nara, Futoshi; Nagayama, Takahiro

    2017-01-15

    It has been elucidated that mineralocorticoid receptor antagonists reduce mortality in patients with congestive heart failure and post-acute myocardial infarction. A direct inhibition of aldosterone synthase (CYP11B2) is also expected to have therapeutic benefits equal in quality to mineralocorticoid receptor antagonists in terms of reducing mineralocorticoid receptor signaling. Therefore, we have screened our chemical libraries and identified a novel and potent aldosterone synthase inhibitor, 2,2,2-trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-y}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol (compound 1), by lead optimization. Pharmacological properties of compound 1 were examined in in vitro cell-based assays and an in vivo mouse model of pressure-overload hypertrophy by transverse aortic constriction (TAC). Compound 1 showed potent CYP11B2 inhibition against human and mouse enzymes (IC50; 0.003μM and 0.096μM, respectively) in a cell-based assay. The oral administration of 0.06% compound 1 in the food mixture of a mouse TAC model significantly reduced the plasma aldosterone level and ameliorated mortality rate. This study is the first to demonstrate that a CYP11B2 inhibitor improved survival rates of heart failure induced by pressure-overload in mice. The treatment of 0.06% compound 1 did not elevate plasma potassium level in this model, although further evaluation of hyperkalemia is needed. These results suggest that compound 1 can be developed as a promising oral CYP11B2 inhibitor for pharmaceutical applications. Compound 1 could also be a useful compound for clarifying the role of aldosterone in cardiac hypertrophy.

  6. Association of Aldosterone Synthase Polymorphism (CYP11B2 -344T>C) and Genetic Ancestry with Atrial Fibrillation and Serum Aldosterone in African Americans with Heart Failure

    PubMed Central

    Bress, Adam; Han, Jin; Patel, Shitalben R.; Desai, Ankit A.; Mansour, Ibrahim; Groo, Vicki; Progar, Kristin; Shah, Ebony; Stamos, Thomas D.; Wing, Coady; Garcia, Joe G. N.; Kittles, Rick; Cavallari, Larisa H.

    2013-01-01

    The objective of this study was to examine the extent to which aldosterone synthase genotype (CYP11B2) and genetic ancestry correlate with atrial fibrillation (AF) and serum aldosterone in African Americans with heart failure. Clinical data, echocardiographic measurements, and a genetic sample for determination of CYP11B2 -344T>C (rs1799998) genotype and genetic ancestry were collected from 194 self-reported African Americans with chronic, ambulatory heart failure. Genetic ancestry was determined using 105 autosomal ancestry informative markers. In a sub-set of patients (n = 126), serum was also collected for determination of circulating aldosterone. The CYP11B2 −344C allele frequency was 18% among the study population, and 19% of patients had AF. Multiple logistic regression revealed that the CYP11B2 −344CC genotype was a significant independent predictor of AF (OR 12.7, 95% CI 1.60–98.4, p = 0.0150, empirical p = 0.011) while holding multiple clinical factors, left atrial size, and percent European ancestry constant. Serum aldosterone was significantly higher among patients with AF (p = 0.036), whereas increased West African ancestry was inversely correlated with serum aldosterone (r = −0.19, p = 0.037). The CYP11B2 −344CC genotype was also overrepresented among patients with extreme aldosterone elevation (≥90th percentile, p = 0.0145). In this cohort of African Americans with chronic ambulatory heart failure, the CYP11B2 −344T>C genotype was a significant independent predictor of AF while holding clinical, echocardiographic predictors, and genetic ancestry constant. In addition, increased West African ancestry was associated with decreased serum aldosterone levels, potentially providing an explanation for the lower risk for AF observed among African Americans. PMID:23936266

  7. Effect of ions of potassium and lithium on NO synthase expression in the human adrenal cortex.

    PubMed

    Kovzun, E I; Lukashenya, O S; Pushkarev, V M; Mikosha, A S; Tron'ko, N D

    2014-01-01

    The expression of endothelial and inducible NO synthase in the human adrenal glands was studied under a change in the concentration of K(+), which plays a regulatory role in aldosterone secretion. K(+) ions stimulated the expression of both isoforms of NO synthase in the human adrenal cortex. A stimulatory effect of K(+) on NO synthase is probably related to activation of the calmodulin system and potassium-induced translocation of protein kinase C. Lithium produced n inhibitory effect on both isoforms of NO synthase, which suggests that protein kinase C serves a major regulator of expression in the human adrenal glands.

  8. Discovery of Potential Inhibitors of Aldosterone Synthase from Chinese Herbs Using Pharmacophore Modeling, Molecular Docking, and Molecular Dynamics Simulation Studies

    PubMed Central

    Lu, Fang; Qiao, Liansheng; Chen, Xi; Li, Gongyu

    2016-01-01

    Aldosterone synthase (CYP11B2) is a key enzyme for the biosynthesis of aldosterone, which plays a significant role for the regulation of blood pressure. Excess aldosterone can cause the dysregulation of the renin-angiotensin-aldosterone system (RAAS) and lead to hypertension. Therefore, research and development of CYP11B2 inhibitor are regarded as a novel approach for the treatment of hypertension. In this study, the pharmacophore models of CYP11B2 inhibitors were generated and the optimal model was used to identify potential CYP11B2 inhibitors from the Traditional Chinese Medicine Database (TCMD, Version 2009). The hits were further refined by molecular docking and the interactions between compounds and CYP11B2 were analyzed. Compounds with high Fitvalue, high docking score, and expected interactions with key residues were selected as potential CYP11B2 inhibitors. Two most promising compounds, ethyl caffeate and labiatenic acid, with high Fitvalue and docking score were reserved for molecular dynamics (MD) study. All of them have stability of ligand binding which suggested that they might perform the inhibitory effect on CYP11B2. This study provided candidates for novel drug-like CYP11B2 inhibitors by molecular simulation methods for the hypertension treatment. PMID:27781210

  9. Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension

    PubMed Central

    Chen, Dong-Rui; Ruan, Cheng-Chao; Xu, Jian-Zhong; Chen, Jing; Wu, Yong-Jie; Ma, Yu; Zhu, Ding-Liang; Gao, Ping-Jin

    2016-01-01

    The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement. PMID:27661131

  10. [Prothrombotic aldosterone action--a new side of the hormone].

    PubMed

    Gromotowicz, Anna; Osmólska, Urszula; Mantur, Maria; Szoka, Piotr; Zakrzeska, Agnieszka; Szemraj, Janusz; Chabielska, Ewa

    2010-10-18

    Recent studies have focused on a new wave of interest in aldosterone due mainly to its growing profile as a local messenger in pathology of the cardiovascular system, rather than its hormonal action. In the last few years strong evidence for a correlation between raised aldosterone level and haemostasis disturbances leading to increased risk of cardiovascular events has been provided. It has been demonstrated that aldosterone contributes to endothelial dysfunction, fibrinolytic disorders and oxidative stress augmentation. It was also shown that chronic aldosterone treatment results in enhanced experimental arterial thrombosis. Our study in a venous model of thrombosis in normotensive rats confirmed that even a short-lasting increase in aldosterone level intensified thrombus formation. One-hour aldosterone infusion shortened bleeding time; increased platelet adhesion to collagen; reduced tissue factor, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor; and increased plasminogen activator plasma level. A fall in plasma nitric oxide metabolite concentration with a decrease in aortic nitric oxide synthase mRNA level was also observed. Moreover, aldosterone increased hydrogen peroxide and malonyl dialdehyde plasma concentration and augmented NADPH oxidase and superoxide dismutase aortic expression. Therefore, the mechanism of aldosterone prothrombotic action is multiple and involves primary haemostasis activation, procoagulative and antifibrinolytic action, NO bioavailability impairment and oxidative stress augmentation. The effects of aldosterone were not fully abolished by mineralocorticoid receptor blockade, suggesting the involvement of alternative mechanisms in the prothrombotic aldosterone action.

  11. In vivo regulation of gene expression of enzymes controlling aldosterone synthesis in rat adrenal.

    PubMed

    LeHoux, J G; Tremblay, A

    1992-12-01

    We studied the effect of alterations in the intake of sodium and potassium as well as changes in circulating adrenocorticotropin (ACTH) on the expression of the two rate-limiting systems of aldosterone formation in the rat. Low sodium and high potassium intake promoted time-dependent increases in the zona glomerulosa cytochrome P450scc (P450scc) and cytochrome P450c11 (P450c11) protein and mRNA levels, but no changes were found in the zona fasciculata-reticularis. In addition, these responses were associated with markedly elevated transcriptional activities. To further define the contribution of P450c11 and P450c18 (aldosterone synthase) in response to these differing intakes, we evaluated their mRNA levels using gene-specific oligonucleotide probes. P450c18 mRNA was restricted to the zona glomerulosa, whereas P450c11 mRNA was detected in both zona glomerulosa and zona fasciculata-reticularis. Furthermore, only P450c18 mRNA was induced by both low sodium or high potassium intake, as P450c11 mRNA levels remained unchanged. Captopril, an inhibitor of angiotensin-I converting enzyme, abolished the enhancing effects of the low sodium regimen on P450scc and P450c18 mRNA levels. Captopril also suppressed the augmentation of P450c18 mRNA observed with potassium supplementation but had no effect on P450scc mRNA levels. When the hypocholesterolemic drug 4-aminopyrazolopyrimidine (4-APP) was administered to rats for 3 consecurive days, both the level of plasma ACTH and the adrenal content of mRNA encoding P450scc increased 24 h post final injection. The coadministration of dexamethasone with 4-APP prevented these increases. In contrast, the mRNA content of P450c11 remained at control levels. In conclusion, this work demonstrates that variations in the intake of sodium and potassium act on the expression of the CYP11B2 gene, but not on that of the CYP11B1 gene. Moreover angiotensin-II (A-II) is an important factor in this mechanism of action. Both ions also enhance the

  12. Subchronic treatment with aldosterone induces depression-like behaviours and gene expression changes relevant to major depressive disorder.

    PubMed

    Hlavacova, Natasa; Wes, Paul D; Ondrejcakova, Maria; Flynn, Marianne E; Poundstone, Patricia K; Babic, Stanislav; Murck, Harald; Jezova, Daniela

    2012-03-01

    The potential role of aldosterone in the pathophysiology of depression is unclear. The aim of this study was to test the hypothesis that prolonged elevation of circulating aldosterone induces depression-like behaviour accompanied by disease-relevant changes in gene expression in the hippocampus. Subchronic (2-wk) treatment with aldosterone (2 μg/100 g body weight per day) or vehicle via subcutaneous osmotic minipumps was used to induce hyperaldosteronism in male rats. All rats (n = 20/treatment group) underwent a modified sucrose preference test. Half of the animals from each treatment group were exposed to the forced swim test (FST), which served both as a tool to assess depression-like behaviour and as a stress stimulus. Affymetrix microarray analysis was used to screen the entire rat genome for gene expression changes in the hippocampus. Aldosterone treatment induced an anhedonic state manifested by decreased sucrose preference. In the FST, depressogenic action of aldosterone was manifested by decreased latency to immobility and increased time spent immobile. Aldosterone treatment resulted in transcriptional changes of genes in the hippocampus involved in inflammation, glutamatergic activity, and synaptic and neuritic remodelling. Furthermore, aldosterone-regulated genes substantially overlapped with genes affected by stress in the FST. This study demonstrates the existence of a causal relationship between the hyperaldosteronism and depressive behaviour. In addition, aldosterone treatment induced changes in gene expression that may be relevant to the aetiology of major depressive disorder. Subchronic treatment with aldosterone represents a new animal model of depression, which may contribute to the development of novel targets for the treatment of depression.

  13. Effect of aldosterone on BK channel expression in mammalian cortical collecting duct.

    PubMed

    Estilo, Genevieve; Liu, Wen; Pastor-Soler, Nuria; Mitchell, Phillip; Carattino, Marcelo D; Kleyman, Thomas R; Satlin, Lisa M

    2008-09-01

    Apical large-conductance Ca(2+)-activated K(+) (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K(+) secretion. Dietary K(+) loading for 10-14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K(+) secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922-F932, 2005). To test whether this adaptation was mediated by a K(+)-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na(+) (LS) or high-Na(+) (HS) diet for 7-10 days to alter circulating levels of aldosterone but not serum K(+) concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, alpha-splice variants, beta-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na(+) (J(Na)) and K(+) (J(K)) transport by microperfusion; kidneys were processed for immunolocalization of BK alpha-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na(+) and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK alpha-, beta(2)-, and beta(4)-subunit mRNA and localization of immunodetectable alpha-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from approximately 1 to 5 nl.min(-1).mm(-1), the increase in J(Na) was greater in LS vs. HS rabbits, yet the flow-stimulated increase in J(K) was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K(+) loading.

  14. Role of ACTH and Other Hormones in the Regulation of Aldosterone Production in Primary Aldosteronism

    PubMed Central

    El Ghorayeb, Nada; Bourdeau, Isabelle; Lacroix, André

    2016-01-01

    aldosteronism, a chimeric CYP11B2 becomes regulated by ACTH activating its chimeric CYP11B1 promoter of aldosterone synthase in bilateral adrenal fasciculate-like hyperplasia. This review will focus on the role of ACTH on excess aldosterone secretion in PA with particular focus on the aberrant expression of MC2R in comparison with other aberrant ligands and their GPCRs in this frequent pathology. PMID:27445975

  15. In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

    PubMed

    Lucas, Simon; Heim, Ralf; Ries, Christina; Schewe, Katarzyna E; Birk, Barbara; Hartmann, Rolf W

    2008-12-25

    Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

  16. Modulation of cytochromes P450 with xanthone-based molecules: from aromatase to aldosterone synthase and steroid 11β-hydroxylase inhibition.

    PubMed

    Gobbi, Silvia; Hu, Qingzhong; Negri, Matthias; Zimmer, Christina; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Bisi, Alessandra

    2013-02-28

    Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

  17. Pharmacological treatment of aldosterone excess.

    PubMed

    Deinum, Jaap; Riksen, Niels P; Lenders, Jacques W M

    2015-10-01

    Primary aldosteronism, caused by autonomous secretion of aldosterone by the adrenals, is estimated to account for at least 5% of hypertension cases. Hypertension explains the considerable cardiovascular morbidity caused by aldosteronism only partly, calling for specific anti-aldosterone drugs. The pharmacology of aldosterone is complex due to high homology with other steroids, the resemblance of steroid receptors, and the common pathways of steroid synthesis. Classically, pharmacological treatment of aldosteronism relied on the mineralocorticoid receptor (MR) antagonist spironolactone, which is highly effective, but causes considerable, mainly sexual side-effects due to limited selectivity for the MR. New agents have been developed or are being developed that aim at higher selectivity for MR antagonists (eplerenone, dihydropyridine-derived calcium channel blockers (CCB)), or inhibition of aldosterone synthesis. Eplerenone is less potent than spironolactone, but causes fewer adverse effects due to its selectivity for the MR. Non-steroidal MR antagonists have been developed from dihydropyridine CCBs, having lost their CCB activity and being highly selective for the MR. The first clinical studies with these drugs are underway. Aldosterone synthase inhibitors are an attractive alternative, but are prone to interference with cortisol synthesis due to the inhibition of 11-β-hydroxylation, an essential step in both cortisol and aldosterone synthesis, and accumulation of mineralocorticoid precursors. In coming years clinical research will provide the answers as to which drugs and strategies to treat high-aldosterone states are the most effective.

  18. Primary Aldosteronism

    MedlinePlus

    ... Endocrinology Find an Endocrinologist Value of an Endocrinologist Learn About Clinical Trials Keep Your Body in Balance › Primary Aldosteronism Fact Sheet Primary Aldosteronism March 2012 Download PDFs English Espanol Editors Paul Stewart, MD, FRCP William Young, ...

  19. Aldosterone regulates cellular turnover and mitogen-activated protein kinase family expression in the neonatal rat kidney.

    PubMed

    Yim, Hyung Eun; Yoo, Kee Hwan; Bae, In Sun; Jang, Gi Young; Hong, Young Sook; Lee, Joo Won

    2009-06-01

    Growing evidence indicates that aldosterone is a potent mitogenic signal regulating genes involved in antiapoptosis, cell proliferation and growth. We investigated the role of endogenous aldosterone in renal development, cell proliferation and apoptosis, and mitogen-activated protein kinase (MAPK) family expression. Newborn rats were treated with either spironolactone (200 mg/kg/d) in olive oil or only olive oil for 7 days. TUNEL assay and proliferating cell nuclear antigen (PCNA) stain were performed on kidney sections. Immunoblots, immunohistochemical (IHC) stain, and reverse transcriptase-PCR for MAPKs were performed. PCNA-positive proliferating cells decreased and apoptotic cells increased significantly with spironolactone (P < 0.05). In the spironolactone-treated group, c-jun N-terminal kinase (JNK)-2 expression increased, whereas extracellular signal regulated kinase (ERK)-2 and p38 expressions decreased in immunoblots (P < 0.05) and IHC stain. ERK-2 and p38 mRNA expressions increased in the spironolactone-treated group (P < 0.05). This study demonstrates that aldosterone blockade in the developing kidney decreases cellular proliferation, increases apoptosis, and modulates the expressions of JNK-2, ERK-2, and p38. Aldosterone possibly participates in renal development and MAPK family may serve as, in part, the signaling intermediate through the mineralocorticoid receptor (MR) in the developing kidney. J. Cell. Physiol. 219: 724-733, 2009. (c) 2009 Wiley-Liss, Inc.

  20. Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues

    PubMed Central

    Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G.; Yeo, Giles S.H.; Brown, Morris J.

    2016-01-01

    Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)–mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary

  1. Molecular Heterogeneity in Aldosterone-Producing Adenomas

    PubMed Central

    Nanba, Kazutaka; Chen, Andrew X.; Omata, Kei; Vinco, Michelle; Giordano, Thomas J.; Else, Tobias; Hammer, Gary D.

    2016-01-01

    Context: The use of next-generation sequencing has resulted in the identification of recurrent somatic mutations underlying primary aldosteronism (PA). However, significant gaps remain in our understanding of the relationship between tumor aldosterone synthase (CYP11B2) expression and somatic mutation status. Objective: The objective of the study was to investigate tumor CYP11B2 expression and somatic aldosterone-driver gene mutation heterogeneity. Methods: Fifty-one adrenals from 51 PA patients were studied. Immunohistochemistry for CYP11B2 was performed. Aldosterone-producing adenomas with intratumor CYP11B2 heterogeneity were analyzed for mutation status using targeted next-generation sequencing. DNA was isolated from CYP11B2-positive, CYP11B2-negative, and adjacent normal areas from formalin-fixed, paraffin-embedded sections. Results: Of 51 adrenals, seven (14 %) showed distinct heterogeneity in CYP11B2 by immunohistochemistry, including six adenomas with intratumor heterogeneity and one multinodular hyperplastic adrenal with both CYP11B2-positive and -negative nodules. Of the six adrenocortical adenomas with CYP11B2 heterogeneity, three had aldosterone-regulating mutations (CACNA1D p.F747C, KCNJ5 p.L168R, ATP1A1 p.L104R) only in CYP11B2-positive regions, and one had two different mutations localized to two histologically distinct CYP11B2-positive regions (ATP2B3 p.L424_V425del, KCNJ5 p.G151R). Lastly, one adrenal with multiple CYP11B2-expressing nodules showed different mutations in each (CACNA1D p.F747V and ATP1A1 p.L104R), and no mutations were identified in CYP11B2-negative nodule or adjacent normal adrenal. Conclusions: Adrenal tumors in patients with PA can demonstrate clear heterogeneity in CYP11B2 expression and somatic mutations in driver genes for aldosterone production. These findings suggest that aldosterone-producing adenoma tumorigenesis can occur within preexisting nodules through the acquisition of somatic mutations that drive aldosterone

  2. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands.

    PubMed

    Nishimoto, Koshiro; Tomlins, Scott A; Kuick, Rork; Cani, Andi K; Giordano, Thomas J; Hovelson, Daniel H; Liu, Chia-Jen; Sanjanwala, Aalok R; Edwards, Michael A; Gomez-Sanchez, Celso E; Nanba, Kazutaka; Rainey, William E

    2015-08-18

    Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

  3. Aberrant gonadotropin-releasing hormone receptor (GnRHR) expression and its regulation of CYP11B2 expression and aldosterone production in adrenal aldosterone-producing adenoma (APA).

    PubMed

    Nakamura, Yasuhiro; Hattangady, Namita G; Ye, Ping; Satoh, Fumitoshi; Morimoto, Ryo; Ito-Saito, Takako; Sugawara, Akira; Ohba, Koji; Takahashi, Kazuhiro; Rainey, William E; Sasano, Hironobu

    2014-03-25

    Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA.

  4. Understanding primary aldosteronism: impact of next generation sequencing and expression profiling

    PubMed Central

    Monticone, Silvia; Else, Tobias; Mulatero, Paolo; Williams, Tracy A.; Rainey, William E.

    2014-01-01

    Primary aldosteronism (PA) encompasses a broad, heterogeneous group of disorders including both sporadic and familial forms (familial hyperaldosteronism type I, II and III). PA is the most common form of secondary hypertension and associated with a higher rate of cardiovascular complications, compared to essential hypertension. Despite significant progress in the diagnosis and management of PA, until recently the molecular mechanisms leading to inappropriate aldosterone production were largely unknown. The introduction of next-generation sequencing has had a profound impact on the field of human genetics and has given new insight in the molecular determinants that lead to both sporadic and familial forms of PA. Here we review the recent progress towards understanding of the genetic and molecular mechanisms leading to autonomous aldosterone production in PA. PMID:25240470

  5. Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

    PubMed

    Dave, Kashyap; Punekar, Narayan S

    2011-09-10

    Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.

  6. [Primary aldosteronism].

    PubMed

    Amar, Laurence

    2015-06-01

    Primary aldosteronism affects 6% of hypertensive patients. The diagnosis should be suspected in any patient with severe or resistant hypertension or hypertension associated with hypokalemia. The screening test consists on the assessment of the aldosterone to renin ratio. In case of an elevated ratio, the diagnosis of primary aldosteronism is confirmed by either elevated concentrations of basal plasma and/or urinary aldosterone or absence of suppression of aldosterone during dynamic test (including the saline infusion test). CT aims to ensure the absence of adrenal carcinoma and to study the morphology of the adrenals. The unilateral or bilateral type of aldosterone secretion is based on the realization of an adrenal venous sampling. When the hypersecretion is unilateral, the treatment consists of adrenalectomy leading to cure of hypertension in 42% of cases, improvement in 40% of cases. For patient with bilateral disease or who don't want to undergo surgery, treatment is based on spironolactone usually at doses of 25 or 50 mg in combination with other antihypertensives drugs such as diuretics or calcium channel blockers.

  7. Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

    PubMed

    Bai, Juan; Chow, Billy K C

    2017-04-01

    Secretin (SCT) and its receptor (SCTR) are important in fluid regulation at multiple levels via the modulation of expression and translocation of renal aquaporin 2 and functions of central angiotensin II (ANGII). The functional interaction of SCT with peripheral ANGII, however, remains unknown. As the ANGII-aldosterone axis dominates the regulation of renal epithelial sodium channel (ENaC) function, we therefore tested whether SCT/SCTR can regulate sodium homeostasis via the renin-angiotensin-aldosterone system. SCTR-knockout (SCTR(-/-)) mice showed impaired aldosterone synthase (CYP11B2) expression and, consequently, aldosterone release upon intraperitoneal injection of ANGII. Endogenous ANGII production induced by dietary sodium restriction was higher in SCTR(-/-) than in C57BL/6N [wild-type (WT)] mice, but CYP11B2 and aldosterone synthesis were not elevated. Reduced accumulation of cholesteryl ester-the precursor of aldosterone-was observed in adrenal glands of SCTR(-/-) mice that were fed a low-sodium diet. Absence of SCTR resulted in elevated basal transcript levels of adrenal CYP11B2 and renal ENaCs. Although transcript and protein levels of ENaCs were similar in WT and SCTR(-/-) mice under sodium restriction, ENaCs in SCTR(-/-) mice were less sensitive to amiloride hydrochloride. In summary, the SCT/SCTR axis is involved in aldosterone precursor uptake, and the knockout of SCTR results in defective aldosterone biosynthesis/release and altered sensitivity of ENaCs to amiloride.-Bai, J., Chow, B. K. C. Secretin is involved in sodium conservation through the renin-angiotensin-aldosterone system.

  8. Osmotic stress regulates mineralocorticoid receptor expression in a novel aldosterone-sensitive cortical collecting duct cell line.

    PubMed

    Viengchareun, Say; Kamenicky, Peter; Teixeira, Marie; Butlen, Daniel; Meduri, Geri; Blanchard-Gutton, Nicolas; Kurschat, Christine; Lanel, Aurélie; Martinerie, Laetitia; Sztal-Mazer, Shoshana; Blot-Chabaud, Marcel; Ferrary, Evelyne; Cherradi, Nadia; Lombès, Marc

    2009-12-01

    Aldosterone effects are mediated by the mineralocorticoid receptor (MR), a transcription factor highly expressed in the distal nephron. Given that MR expression level constitutes a key element controlling hormone responsiveness, there is much interest in elucidating the molecular mechanisms governing MR expression. To investigate whether hyper- or hypotonicity could affect MR abundance, we established by targeted oncogenesis a novel immortalized cortical collecting duct (CCD) cell line and examined the impact of osmotic stress on MR expression. KC3AC1 cells form domes, exhibit a high transepithelial resistance, express 11beta-hydroxysteroid dehydrogenase 2 and functional endogenous MR, which mediates aldosterone-stimulated Na(+) reabsorption through the epithelial sodium channel activation. MR expression is tightly regulated by osmotic stress. Hypertonic conditions induce expression of tonicity-responsive enhancer binding protein, an osmoregulatory transcription factor capable of binding tonicity-responsive enhancer response elements located in MR regulatory sequences. Surprisingly, hypertonicity leads to a severe reduction in MR transcript and protein levels. This is accompanied by a concomitant tonicity-induced expression of Tis11b, a mRNA-destabilizing protein that, by binding to the AU-rich sequences of the 3'-untranslated region of MR mRNA, may favor hypertonicity-dependent degradation of labile MR transcripts. In sharp contrast, hypotonicity causes a strong increase in MR transcript and protein levels. Collectively, we demonstrate for the first time that optimal adaptation of CCD cells to changes in extracellular fluid composition is accompanied by drastic modification in MR abundance via transcriptional and posttranscriptional mechanisms. Osmotic stress-regulated MR expression may represent an important molecular determinant for cell-specific MR action, most notably in renal failure, hypertension, or mineralocorticoid resistance.

  9. Dietary salt modulates the sodium chloride cotransporter expression likely through an aldosterone-mediated WNK4-ERK1/2 signaling pathway.

    PubMed

    Lai, Lingyun; Feng, Xiuyan; Liu, Defeng; Chen, Jing; Zhang, Yiqian; Niu, Bowen; Gu, Yong; Cai, Hui

    2012-03-01

    WNK is a serine/threonine kinase. Mutation in WNK1 or WNK4 kinase results in pseudohypoaldosteronism type II (PHA II) featuring hypertension, hyperkalemia and metabolic acidosis. Sodium chloride cotransporter (NCC) is known to be regulated by phosphorylation and trafficking. Dietary salt and hormonal stimulation, such as aldosterone, also affect the regulation of NCC. We have previously reported that WNK4 inhibits NCC protein expression. To determine whether dietary salt affects NCC abundance through WNK4-mediated mechanism, we investigated the effects of dietary salt change with or without aldosterone infusion (1 mg/kg/day) on NCC and WNK4 expression in rats. We found that high-salt (HS, 4% NaCl) diet significantly inhibits NCC mRNA expression and protein abundance while enhancing WNK4 mRNA and protein expression, whereas low-salt (LS, 0.07% NaCl) diet increases NCC mRNA expression and protein abundance while reducing WNK4 expression. We also found that aldosterone infusion in HS-fed rats increases NCC mRNA expression and protein abundance, but decreases WNK4 expression. Administration with spironolactone (0.1 g/kg/day) in LS-fed rats decreases NCC mRNA expression and protein abundance while increasing WNK4 expression. We further showed that ERK1/2 phosphorylation was increased in HS-fed rats, but decreased in LS-fed rats. In HEK293 cells, over-expressed WNK4 increases ERK1/2 phosphorylation, whereas knockdown of WNK4 expression decreases ERK1/2 phosphorylation. Aldosterone treatment for 3 h decreases ERK1/2 phosphorylation. These data suggest that dietary salt change affects NCC protein abundance in an aldosterone-dependent mechanism likely via the WNK4-ERK1/2-mediated pathway.

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

    PubMed

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

    2003-12-07

    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.

  11. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    PubMed

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

  12. Structural, functional, and molecular alterations produced by aldosterone plus salt in rat heart: association with enhanced serum and glucocorticoid-regulated kinase-1 expression.

    PubMed

    Martín-Fernández, Beatriz; de las Heras, Natalia; Miana, María; Ballesteros, Sandra; Delgado, Carmen; Song, Su; Hintze, Thomas; Cachofeiro, Victoria; Lahera, Vicente

    2011-01-01

    We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg[middle dot]kg-1[middle dot]d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg[middle dot]kg-1[middle dot]d-1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1[beta], p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.

  13. Aldosterone blood test

    MedlinePlus

    ... hypotension) Aldosterone is a hormone released by the adrenal glands . It helps the body regulate blood pressure. Aldosterone ... may be due to Bartter syndrome (extremely rare) Adrenal glands release too much aldosterone hormone ( primary hyperaldosteronism - usually ...

  14. Identification of a Dolabellane Type Diterpene Synthase and other Root-Expressed Diterpene Synthases in Arabidopsis

    PubMed Central

    Wang, Qiang; Jia, Meirong; Huh, Jung-Hyun; Muchlinski, Andrew; Peters, Reuben J.; Tholl, Dorothea

    2016-01-01

    Arabidopsis thaliana maintains a complex metabolism for the production of secondary or specialized metabolites. Such metabolites include volatile and semivolatile terpenes, which have been associated with direct and indirect defensive activities in flowers and leaves. In comparison, the structural diversity and function of terpenes in Arabidopsis roots has remained largely unexplored despite a substantial number of root-expressed genes in the Arabidopsis terpene synthase (TPS) gene family. We show that five root-expressed TPSs of an expanded subfamily-a type clade in the Arabidopsis TPS family function as class I diterpene synthases that predominantly convert geranylgeranyl diphosphate (GGPP) to different semi-volatile diterpene products, which are in part detectable at low levels in the ecotypes Columbia (Col) and Cape Verde Island (Cvi). The enzyme TPS20 produces a macrocyclic dolabellane diterpene alcohol and a dolabellane-related diterpene olefin named dolathaliatriene with a so far unknown C6-C11 bicyclic scaffold besides several minor olefin products. The TPS20 compounds occur in all tissues of Cvi but are absent in the Col ecotype because of deletion and substitution mutations in the Col TPS20 sequence. The primary TPS20 diterpene products retard the growth of the root rot pathogen Pythium irregulare but only at concentrations exceeding those in planta. Together, our results demonstrate that divergence and pseudogenization in the Arabidopsis TPS gene family allow for structural plasticity in diterpene profiles of above- and belowground tissues. PMID:27933080

  15. Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

    PubMed Central

    Eldar-Finkelman, H; Argast, G M; Foord, O; Fischer, E H; Krebs, E G

    1996-01-01

    In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8816781

  16. StAR expression and the long-term aldosterone response to high-potassium diet in Wistar-Kyoto and spontaneously hypertensive rats.

    PubMed

    Peters, Barbara; Teubner, Philipp; Clausmeyer, Susanne; Puschner, Tanja; Maser-Gluth, Christiane; Wrede, Hans-Josef; Kränzlin, Bettina; Peters, Jörg

    2007-01-01

    ANG II and potassium are known to increase steroidogenic acute regulatory protein (StAR) levels. However, a corresponding increase in StAR mRNA levels has so far been observed only in response to ANG II. We therefore studied the regulation of adrenal StAR mRNA expression in the context of dietary potassium-stimulated aldosterone production. Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were fed a diet containing either 1 or 4% KCl for 5 days. The high-potassium diet increased StAR mRNA levels within the zona glomerulosa in both strains, as demonstrated by in situ hybridization. However, aldosterone production increased in WKY but not in SHR (WKY: from 22.8 +/- 4.8 to 137 +/- 25 ng/100 ml, P < 0.001, vs. SHR: from 29 +/- 3.8 to 51 +/- 10.2 ng/100 ml, not significant). This increase was associated with an increase in Cyp11b2 mRNA levels in WKY (3-fold; P < 0.001) but not in SHR. In both strains, the 4% KCl diet was associated with increased plasma renin-independent aldosterone production, as indicated by the marked increase of the aldosterone-to-renin ratios (from 1.4 +/- 0.3 to 9 +/- 3 in WKY and from 3 +/- 1 to 14 +/- 5 in SHR; P < 0.002). We conclude that an increase of StAR mRNA levels within the outer cortex is involved in the long-term adrenal response to potassium. This increase alone is not sufficient to increase aldosterone production in the presence of normal Cyp11b2 mRNA levels.

  17. [Cloning, expression and charaterization of chalcone synthase from Saussurea medusa].

    PubMed

    Xia, Fang; Li, Houhua; Fu, Chunxiang; Yu, Zhenzhen; Xu, Yanjun; Zhao, Dexiu

    2011-09-01

    A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1313 bp, containing an open reading frame (1170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

  18. Manipulation of pulmonary prostacyclin synthase expression prevents murine lung cancer.

    PubMed

    Keith, Robert L; Miller, York E; Hoshikawa, Yasushi; Moore, Mark D; Gesell, Tracy L; Gao, Bifeng; Malkinson, Alvin M; Golpon, Heiko A; Nemenoff, Raphael A; Geraci, Mark W

    2002-02-01

    Inhibition of cyclooxygenase (COX) activity decreases eicosanoid production and prevents lung cancer in animal models. Prostaglandin (PG) I(2) (PGI(2), prostacyclin) is a PGH(2) metabolite with anti-inflammatory, antiproliferative, and antimetastatic properties. The instability of PGI(2) has limited its evaluation in animal models of cancer. We hypothesized that pulmonary overexpression of prostacyclin synthase may prevent the development of murine lung tumors. Transgenic mice with selective pulmonary prostacyclin synthase overexpression were exposed to two distinct carcinogenesis protocols: an initiation/promotion model and a simple carcinogen model. The transgenic mice exhibited significantly reduced lung tumor multiplicity (tumor number) in proportion to transgene expression, a dose-response effect. Moreover, the highest expressing mice demonstrated reduced tumor incidence. To investigate the mechanism for protection, we evaluated PG levels and inflammatory responses. At the time of sacrifice following one carcinogenesis model, the transgenics exhibited only an increase in 6-keto-PGF(1alpha), not a decrease in PGE(2). Thus, elevated PGI(2) levels and not decreased PGE(2) levels appear to be necessary for the chemopreventive effects. When exposed to a single dose of butylated hydroxytoluene, transgenic mice exhibited a survival advantage; however, reduction in alveolar inflammatory response was not observed. These studies demonstrate that manipulation of PG metabolism downstream from COX produces even more profound lung cancer reduction than COX inhibition alone and could be the basis for new approaches to understanding the pathogenesis and prevention of lung cancer.

  19. Expression of human hyaluronan synthases in response to external stimuli.

    PubMed Central

    Jacobson, A; Brinck, J; Briskin, M J; Spicer, A P; Heldin, P

    2000-01-01

    In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins. PMID:10794710

  20. Renin-angiotensin-aldosterone system inhibitors improve membrane stability and change gene-expression profiles in dystrophic skeletal muscles.

    PubMed

    Chadwick, Jessica A; Bhattacharya, Sayak; Lowe, Jeovanna; Weisleder, Noah; Rafael-Fortney, Jill A

    2017-02-01

    Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

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

    PubMed

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

    2010-03-25

    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.

  2. Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

    PubMed

    Buldreghini, E; Hamada, A; Macrì, M L; Amoroso, S; Boscaro, M; Lenzi, A; Agarwal, A; Balercia, G

    2014-12-01

    In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46 ± 0.38 versus 7.06 ± 0.56, P = 0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

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

    PubMed

    Cedergren, J; Forslund, T; Sundqvist, T; Skogh, T

    2002-10-01

    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 micro mol/l, P = 0.008), whereas a slight increase in l-citrulline (33 +/- 11 versus 26 +/- 9 micro mol/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 micro mol/l, P = 0.054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis.

  4. Both TASK-3 and TREK-1 two-pore loop K channels are expressed in H295R cells and modulate their membrane potential and aldosterone secretion.

    PubMed

    Brenner, Tanja; O'Shaughnessy, Kevin M

    2008-12-01

    The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to K(+) ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of Galpha(q) into H295R cells (GTPase-deficient Galpha(q)-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current.

  5. Evolution and Expression of Tandem Duplicated Maize Flavonol Synthase Genes

    PubMed Central

    Falcone Ferreyra, María Lorena; Casas, María Isabel; Questa, Julia Irene; Herrera, Andrea Lorena; DeBlasio, Stacy; Wang, Jing; Jackson, David; Grotewold, Erich; Casati, Paula

    2012-01-01

    Flavonoids are specialized compounds widely distributed and with diverse functions throughout the plant kingdom and with several benefits for human health. In particular, flavonols, synthesized by flavonol synthase (FLS), protect plants against UV-B radiation and are essential for male fertility in maize and other plants. We have recently characterized a UV-B inducible ZmFLS1, corresponding to the first to be described in monocot plants. Interestingly, the new assembly of the B73 maize genome revealed the presence of a second putative FLS gene (ZmFLS2), with very high identity with ZmFLS1. ZmFLSs expression was analyzed in different maize tissues, and by combining electrophoretic mobility shift assays and transient expression experiments, we show that both genes are direct targets of anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. ZmFLS expression analyses show higher levels of both transcripts in high altitude landraces than inbred lines, and both genes are regulated by UV-B radiation in all lines analyzed. Moreover, the high sequence conservation of the ZmFLS promoters between maize lines suggests that the differences observed in ZmFLS expression are due to allelic variations in the transcription factors that regulate their activities. Finally, we generated pFLS1::FLS1-RFP transgenic plants and analyzed ZmFLS1 expression in different maize tissues; we found that this enzyme is localized in the ER and the perinuclear region. PMID:22654889

  6. Transcriptional regulation of human thromboxane synthase gene expression

    SciTech Connect

    Lee, K.D.; Baek, S.J.; Fleischer, T

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  7. Ectopic expression of ceramide synthase 2 in neurons suppresses neurodegeneration induced by ceramide synthase 1 deficiency

    PubMed Central

    Spassieva, Stefka D.; Ji, Xiaojie; Liu, Ye; Gable, Kenneth; Bielawski, Jacek; Dunn, Teresa M.; Bieberich, Erhard; Zhao, Lihong

    2016-01-01

    Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22–C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypothesis, we found that only LCBs, the substrates common for all of the CerS isoforms, but not ceramides and complex sphingolipids, were restored to the wild-type levels in the Cers2-rescued Cers1 mutant mouse brains. Furthermore, LCBs induced neurite fragmentation in cultured neurons at concentrations corresponding to the elevated levels in the CerS1-deficient brain. The strong association of LCB levels with neuronal survival both in vivo and in vitro suggests high-level accumulation of LCBs is a possible underlying cause of the CerS1 deficiency-induced neuronal death. PMID:27162368

  8. Oxalomalate affects the inducible nitric oxide synthase expression and activity.

    PubMed

    Irace, Carlo; Esposito, Giuseppe; Maffettone, Carmen; Rossi, Antonietta; Festa, Michela; Iuvone, Teresa; Santamaria, Rita; Sautebin, Lidia; Carnuccio, Rosa; Colonna, Alfredo

    2007-03-13

    Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

  9. Aldosterone and the conquest of land.

    PubMed

    Colombo, L; Dalla Valle, L; Fiore, C; Armanini, D; Belvedere, P

    2006-04-01

    The sequence of the phylogenetic events that preceded the appearance of aldosterone in vertebrates is described, starting from the ancestral conversion of cytochrome P450s from oxygen detoxification to xenobiotic detoxification and synthesis of oxygenated endobiotics with useful functions in intercellular signalling, such as steroid hormones. At the end of the Silurian period [438-408 million yr ago, (Mya)], a complete set of cytochrome P450s for corticoid synthesis was presumably already available, except for mitochondrial cytochrome P450c18 or aldosterone synthase encoded by CYP11B2. This gene arose by duplication of the CYP11B gene in the sarcopterygian or lobe-finned fish/tetrapod line after its divergence from the actinopterygian or ray-finned fish line 420 Mya, but before the beginning of the colonization of land by tetrapods in the late Devonian period, around 370 Mya. The fact that aldosterone is already present in Dipnoi, which occupy an evolutionary transition between water- and air-breathing but are fully aquatic, suggests that the role of this steroid was to potentiate the corticoid response to hypoxia, rather than to prevent dehydration out of the water. In terrestrial amphibians, there is no differentiation between the secretion rates and gluco- and mineralocorticoid effects of aldosterone and corticosterone. In sauropsids, plasma aldosterone concentrations are much lower than in amphibians, but regulation of salt/water balance is dependent upon both aldosterone and corticosterone, though sometimes with opposed actions. In terrestrial mammals, aldosterone acquires a specific mineralocorticoid function, because its interaction with the mineralocorticoid receptor is protected by the coexpression of the enzyme 11beta-hydroxysteroid dehydrogenase type 2, which inactivates both cortisol and corticosterone. There is evidence that aldosterone can be also synthesized extra-adrenally in brain neurons and cardiac myocytes, which lack this protection and where

  10. Field performance of transgenic sugarcane expressing isomaltulose synthase.

    PubMed

    Basnayake, Shiromani W V; Morgan, Terrance C; Wu, Luguang; Birch, Robert G

    2012-02-01

    Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3 years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217 mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars. Sugarcane is vegetatively propagated from stem cuttings known as setts. Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field-sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. There was no apparent adverse effect of IM accumulation on vigour assessed by stalk height and diameter or other visual indicators including germination of setts and establishment of stools. There was some inconsistency in IM levels in juice, between samplings of the vegetatively propagated transgenic lines. Until the causes are resolved, it is prudent to selectively propagate from stalks with higher IM levels in the initial vegetative field generations. Pol/Brix ratio allowed rapid identification of lines with high IM levels, using common sugar industry instruments. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial-scale production of IM if higher activity can be engineered in appropriate developmental patterns.

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

    PubMed

    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.

  12. Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

    PubMed Central

    1994-01-01

    A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells. PMID:7806569

  13. Primary aldosteronism and pregnancy.

    PubMed

    Morton, Adam

    2015-10-01

    Primary aldosteronism is the most common cause of secondary hypertension. Less than 50 cases of pregnancy in women with primary aldosteronism have been reported, suggesting the disorder is significantly underdiagnosed in confinement. Accurate diagnosis is complicated by physiological changes in the renin-angiotensin-aldosterone axis in pregnancy, leading to a risk of false negative results on screening tests. The course of primary aldosteronism during pregnancy is highly variable, although overall it is associated with a very high risk of fetal and maternal morbidity and mortality. The optimal management of primary aldosteronism during pregnancy is unclear, with uncertainty regarding the safety of mineralocorticoid antagonists and amiloride, their relative efficacy compared with the antihypertensive medications commonly used during pregnancy, and as to whether prognosis is improved by laparoscopic adrenalectomy where an adrenal adenoma can be demonstrated.

  14. Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases.

    PubMed

    Liu, Benye; Falkenstein-Paul, Hildegard; Schmidt, Werner; Beerhues, Ludger

    2003-06-01

    Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

  15. Transforming growth factor beta1 and aldosterone

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Chang, Albert S.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Purpose of review It is well established that blocking renin-angiotensin II-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. Recent findings TGFbeta1 suppresses adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps from 10% to 300% normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10 % hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. Summary These results identify TGFbeta signaling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism as well as assist in the development of novel therapeutic strategies for hypertension. PMID:25587902

  16. Reduced expression of prostacyclin synthase and nitric oxide synthase in subcutaneous arteries of type 2 diabetic patients.

    PubMed

    Safiah Mokhtar, Siti; M Vanhoutte, Paul; W S Leung, Susan; Imran Yusof, Mohd; Wan Sulaiman, Wan Azman; Zaharil Mat Saad, Arman; Suppian, Rapeah; Ghulam Rasool, Aida Hanum

    2013-01-01

    Diabetic endothelial dysfunction is characterized by impaired endothelium-dependent relaxation. In this study, we measured the expression of endothelial nitric oxide synthase (eNOS), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS), and prostacyclin receptor (IP) in subcutaneous arteries of type-2 diabetic and non-diabetic patients. Subcutaneous arteries were dissected from tissues from seven diabetics (4 males and 3 females) and seven non-diabetics (5 males and 2 females) aged between 18 to 65 years, who underwent lower limb surgical procedures. Diabetics had higher fasting blood glucose compared to non-diabetics, but there were no differences in blood pressure, body mass index and age. Patients were excluded if they had uncontrolled hypertension, previous myocardial infarction, coronary heart disease, renal or hepatic failure and tumor. The relative expression levels of eNOS, COX-1, COX-2, PGIS and IP receptor were determined by Western blotting analysis, normalized with the β-actin level. Increased expression of COX-2 was observed in subcutaneous arteries of diabetics compared to non-diabetics, whereas the expression levels of eNOS and PGIS were significantly lower in diabetics. There were no significant differences in expression levels of COX-1 and IP receptor between the two groups. Immunohistochemical study of subcutaneous arteries showed that the intensities of eNOS and PGIS staining were lower in diabetics, with higher COX-2 staining. In conclusion, type-2 diabetes is associated with higher COX-2 expression, but lower eNOS and PGIS expression in subcutaneous arteries. These alterations may lead to impaired endothelium-dependent vasodilatation, and thus these proteins may be potential targets for protection against the microvascular complications of diabetes.

  17. Molecular cloning, expression, purification, and functional characterization of dammarenediol synthase from Panax ginseng.

    PubMed

    Hu, Wei; Liu, Ning; Tian, Yuhua; Zhang, Lianxue

    2013-01-01

    The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in Rosetta E. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS). Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in Rosetta E. coli and the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides in P. ginseng.

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

    SciTech Connect

    Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J. )

    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.

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

    SciTech Connect

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

    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.

  20. Aldosterone and Renin Test

    MedlinePlus

    ... the kidney (renal vein renin levels). If the value is significantly higher in one side, this indicates where the narrowing of the artery is present. Low aldosterone (hypoaldosteronism) usually occurs as part of ...

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

    PubMed

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

    2012-03-01

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

  2. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    PubMed Central

    Yuan, Guang-Jin; Zhou, Xiao-Rong; Gong, Zuo-Jiong; Zhang, Pin; Sun, Xiao-Mei; Zheng, Shi-Hua

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) expression in the liver. METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65,iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-κB, and TNF-α mRNA expression. CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-κB and TNF-α expression. eNOS activity is reduced, but its mRNA expression is not affected. PMID:16688828

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

    PubMed

    Yamada, Yuuki; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2015-06-01

    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 with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.

  4. EXPRESSION OF THE GEOSMIN SYNTHASE GENE IN THE CYANOBACTERIUM ANABAENA CIRCINALIS AWQC318(1).

    PubMed

    Giglio, Steven; Saint, Christopher P; Monis, Paul T

    2011-12-01

    The occurrence of taste and odor episodes attributed to geosmin continues to trouble water utilities worldwide, and only recently have advances been made in our fundamental understanding of the biochemical and genetic mechanisms responsible for the production of geosmin in microorganisms. For the first time, we have examined the expression of the geosmin synthase gene and corresponding geosmin production by Anabaena circinalis Rabenh. ex Bornet et Flahault AWQC318 under conditions of continuous light illumination and the removal of light as a stimulus and demonstrate that the expression of geosmin synthase appears to be constitutive under these conditions. The decrease in geosmin synthase transcription post maximum cell numbers and stationary phase suggests that a decrease in isoprenoid synthesis may occur before a decrease in the transcription of ribosomal units as the process of cell death is initiated.

  5. LH, progesterone, and TSH can stimulate aldosterone in vitro: a study on normal adrenal cortex and aldosterone producing adenoma.

    PubMed

    Nicolini, G; Balzan, S; Morelli, L; Iacconi, P; Sabatino, L; Ripoli, A; Fommei, E

    2014-05-01

    Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

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

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    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 {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} 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 {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} 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.

  7. Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A Randomized Controlled Trial

    DTIC Science & Technology

    2007-03-01

    14. ABSTRACT: See next page. 15. SUBJECT TERMS Prostate Cancer; Lipid Medtabolism, Clinical Trial; Omega -3 Fatty Acids 16. SECURITY...AD_________________ Award Number: W81XWH-04-1-0296 TITLE: Fish Oil Supplementation and Fatty Acid ...SUBTITLE Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A 5a. CONTRACT NUMBER Randomized

  8. Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase

    SciTech Connect

    Shi, Dashuang Caldovic, Ljubica; Jin, Zhongmin; Yu, Xiaolin; Qu, Qiuhao; Roth, Lauren; Morizono, Hiroki; Hathout, Yetrib; Allewell, Norma M.; Tuchman, Mendel

    2006-12-01

    Expression, crystallization and preliminary X-ray diffraction studies of a novel bifunctional N-acetylglutamate synthase/kinase from X. campestris homologous to vertebrate N-acetylglutamate synthase are reported. A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6{sub 2}22, with unit-cell parameters a = b = 134.60, c = 192.11 Å, and diffract to about 3.0 Å resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method.

  9. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  10. A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine.

    PubMed

    Li, N; Wiesman, Z; Liu, D; Mattoo, A K

    1992-07-20

    1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene. We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli. ACC synthase expressed in E. coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent. Mixing the tomato fruit protein extract with the cell-free extract from transformed E. coli did not affect SAM-dependent inactivation of ACC synthase activity. Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E. coli and their structure-function relationships investigated.

  11. Identification and expression of mutations in the hydroxymethylbilane synthase gene causing acute intermittent porphyria (AIP).

    PubMed Central

    Solis, C.; Lopez-Echaniz, I.; Sefarty-Graneda, D.; Astrin, K. H.; Desnick, R. J.

    1999-01-01

    BACKGROUND: Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (EC 4.3.1.8; HMB-synthase). This disease is characterized by acute, life-threatening neurologic attacks that are precipitated by various drugs, hormones, and other factors. The enzymatic and/or biochemical diagnosis of AIP heterozygotes is problematic; therefore, efforts have focused on the identification of HMB-synthase mutations so that heterozygotes can be identified and educated to avoid the precipitating factors. In Spain, the occurrence of AIP has been reported, but the nature of the HMB-synthase mutations causing AIP in Spanish families has not been investigated. Molecular analysis was therefore undertaken in nine unrelated Spanish AIP patients. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and family members of nine unrelated Spanish families with AIP. The HMB-synthase gene was amplified by long-range PCR and the nucleotide sequence of each exon was determined by cycle sequencing. RESULTS: Three new mutations, a missense, M212V; a single base insertion, g4715insT; and a deletion/insertion, g7902ACT-->G, as well as five previously reported mutations (G111R, R116W, R149X R167W, and R173W) were detected in the Spanish probands. Expression of the novel missense mutation M212V in E. coli revealed that the mutation was causative, having <2% residual activity. CONCLUSIONS: These studies identified the first mutations in the HMB-synthase gene causing AIP in Spanish patients. Three of the mutations were novel, while five previously reported lesions were found in six Spanish families. These findings enable accurate identification and counseling of presymptomatic carriers in these nine unrelated Spanish AIP families and further demonstrate the genetic heterogeneity of mutations causing AIP. Images Fig. 1 PMID:10602775

  12. Placental growth factor mediates aldosterone-dependent vascular injury in mice.

    PubMed

    Jaffe, Iris Z; Newfell, Brenna G; Aronovitz, Mark; Mohammad, Najwa N; McGraw, Adam P; Perreault, Roger E; Carmeliet, Peter; Ehsan, Afshin; Mendelsohn, Michael E

    2010-11-01

    In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

  13. Impaired Wound Induction of 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) Synthase and Altered Stem Development in Transgenic Potato Plants Expressing a DAHP Synthase Antisense Construct.

    PubMed Central

    Jones, J. D.; Henstrand, J. M.; Handa, A. K.; Herrmann, K. M.; Weller, S. C.

    1995-01-01

    Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification. PMID:12228551

  14. Human uroporphyrinogen-III synthase: genomic organization, alternative promoters, and erythroid-specific expression.

    PubMed

    Aizencang, G; Solis, C; Bishop, D F; Warner, C; Desnick, R J

    2000-12-01

    Uroporphyrinogen-III (URO) synthase is the heme biosynthetic enzyme defective in congenital erythropoietic porphyria. The approximately 34-kb human URO-synthase gene (UROS) was isolated, and its organization and tissue-specific expression were determined. The gene had two promoters that generated housekeeping and erythroid-specific transcripts with unique 5'-untranslated sequences (exons 1 and 2A) followed by nine common coding exons (2B to 10). Expression arrays revealed that the housekeeping transcript was present in all tissues, while the erythroid transcript was only in erythropoietic tissues. The housekeeping promoter lacked TATA and SP1 sites, consistent with the observed low level expression in most cells, whereas the erythroid promoter contained GATA1 and NF-E2 sites for erythroid specificity. Luciferase reporter assays demonstrated that the housekeeping promoter was active in both erythroid K562 and HeLa cells, while the erythroid promoter was active only in erythroid cells and its activity was increased during hemin-induced erythroid differentiation. Thus, human URO-synthase expression is regulated during erythropoiesis by an erythroid-specific alternative promoter.

  15. Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli.

    PubMed

    Seo, Yong Bae; Choi, Seong-Seok; Lee, Jong Kyu; Kim, Nan-Hee; Choi, Mi Jin; Kim, Jong-Myoung; Jeong, Tae Hyug; Nam, Soo-Wan; Lim, Han Kyu; Kim, Gun-Do

    2015-11-01

    A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

  16. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    PubMed Central

    2009-01-01

    In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence. PMID:21637458

  17. Molecular cloning and expression profile analysis of three sunflower (Helianthus annuus) diterpene synthase genes.

    PubMed

    Pugliesi, Claudio; Fambrini, Marco; Salvini, Mariangela

    2011-02-01

    ent-Kaurene, a key precursor of gibberellins, is formed by the action of two diterpene synthases (diTPSs), ent-copalyl diphosphate synthase (CPS), and ent-kaurene synthase (KS). The full-length cDNAs of CPS- (HaCPS1L) and KS-like (HaKS2L and HaKS3L) genes were isolated from sunflower. The amino acid sequences of HaCPS1L, HaKS2L, and HaKS3L exhibit structural features and homology to diTPSs of several plant species involved in gibberellin biosynthesis. RT-PCR analysis indicates that the expression of all genes (HaCPS1L, HaKS2L, and HaKS3L) is highly regulated during growth and development. All three diTPSs are preferentially expressed in rapidly growing tissues. HaKS2L is expressed at a much lower level than the other two diTPS genes. During seed development, the high level of both HaCPS1L and HaKS3L transcripts correlated with the period of rapid growth of the embryo. The three diTPS genes are not subjected to feedback regulation by gibberellin activity.

  18. Molecular cloning, expression pattern and comparative analysis of chitin synthase gene B in Spodoptera exigua.

    PubMed

    Kumar, N Senthil; Tang, Bin; Chen, Xiaofei; Tian, Honggang; Zhang, Wenqing

    2008-03-01

    The chitin synthase (CHS) gene B (4781 bp) of Spodoptera exigua (SeCHSB) was cloned by reverse-transcription PCR (RT-PCR) and 3'/5' RACE from the midgut. SeCHSB contains an open reading frame of 4572 nucleotides, encoding a protein of 1523 amino acids with a predicted molecular mass of approximately 174.6 kDa. Alignment of SeCHSB with class B CHSs of other insects showed a high degree of conservation in the putative catalytic domain region. The structure of the SeCHSB gene was analyzed and was found to be the same as that of Manduca sexta CHSB (MsCHSB), including 23 exons and 22 introns but without alternative exons. Southern blot analysis revealed that SeCHSB was a single copy gene and the presence of only two chitin synthase genes in S. exigua. Further investigation indicated that SeCHSB was specifically expressed in the midgut, and its transcript existed constitutively in the midgut from the 3rd instar larval stage to prepupae and reached highest expression on the 1st day of the fifth instar larval stage. These data suggest that SeCHSB is very important in midgut formation and development. Chitin synthase gene comparisons between different classes of insects using software tools revealed some interesting aspects of the similarity and divergence of the gene in the Class Insecta.

  19. Gibberellin Overproduction Promotes Sucrose Synthase Expression and Secondary Cell Wall Deposition in Cotton Fibers

    PubMed Central

    Zhao, Juan; Song, Shui-Qing; Hu, Lin; Zeng, Jian-Yan; Li, Xian-Bi; Hou, Lei; Luo, Ming; Li, De-Mou; Pei, Yan

    2014-01-01

    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. PMID:24816840

  20. Aldosterone Activates NF-κB in the Collecting Duct

    PubMed Central

    Leroy, Valérie; De Seigneux, Sophie; Agassiz, Victor; Hasler, Udo; Rafestin-Oblin, Marie-Edith; Vinciguerra, Manlio; Martin, Pierre-Yves; Féraille, Eric

    2009-01-01

    Besides its classical effects on salt homeostasis in renal epithelial cells, aldosterone promotes inflammation and fibrosis and modulates cell proliferation. The proinflammatory transcription factor NF-κB has been implicated in cell proliferation, apoptosis, and regulation of transepithelial sodium transport. The effect of aldosterone on the NF-κB pathway in principal cells of the cortical collecting duct, a major physiologic target of aldosterone, is unknown. Here, in both cultured cells and freshly isolated rat cortical collecting duct, aldosterone activated the canonical NF-κB signaling pathway, leading to increased expression of several NF-κB–targeted genes (IκBα, plasminogen activator inhibitor 1, monocyte chemoattractant protein 1, IL-1β, and IL-6). Small interfering RNA–mediated knockdown of the serum and glucocorticoid-inducible kinase SGK1, a gene induced early in the response to aldosterone, but not pharmacologic inhibition of extracellular signal–regulated kinase and p38 kinase, attenuated aldosterone-induced NF-κB activation. Pharmacologic antagonism or knockdown of the mineralocorticoid receptor prevented aldosterone-induced NF-κB activity. In addition, activation of the glucocorticoid receptor inhibited the transactivation of NF-κB by aldosterone. In agreement with these in vitro findings, spironolactone prevented NF-κB–induced transcriptional activation observed in cortical collecting ducts of salt-restricted rats. In summary, aldosterone activates the canonical NF-κB pathway in principal cells of the cortical collecting duct by activating the mineralocorticoid receptor and by inducing SGK1. PMID:18987305

  1. Tracking sesamin synthase gene expression through seed maturity in wild and cultivated sesame species--a domestication footprint.

    PubMed

    Pathak, N; Bhaduri, A; Bhat, K V; Rai, A K

    2015-09-01

    Sesamin and sesamolin are the major oil-soluble lignans present in sesame seed, having a wide range of biological functions beneficial to human health. Understanding sesame domestication history using sesamin synthase gene expression could enable delineation of the sesame putative progenitor. This report examined the functional expression of sesamin synthase (CYP81Q1) during capsule maturation (0-40 days after flowering) in three wild Sesamum species and four sesame cultivars. Among the cultivated accessions, only S. indicum (CO-1) exhibited transcript abundance of sesamin synthase along with high sesamin content similar to S. malabaricum, while the other cultivated sesame showed low expression. The sesamin synthase expression analysis, coupled with quantification of sesamin level, indicates that sesamin synthase was not positively favoured during domestication. The sesamin synthase expression pattern and lignan content, along with phylogenetic analysis suggested a close relationship of cultivated sesame and the wild species S. malabaricum. The high genetic identity between the two species S. indicum and S. malabaricum points towards the role of the putative progenitor S. malabaricum in sesame breeding programmes to broaden the genetic base of sesame cultivars. This study emphasises the need to investigate intraspecific and interspecific variation in the primary, secondary and tertiary gene pools to develop superior sesame genotypes.

  2. 2-Methyl-3-buten-2-ol (MBO) synthase expression in Nostoc punctiforme leads to over production of phytols

    PubMed Central

    Gupta, Dinesh; Ip, Tina; Summers, Michael L; Basu, Chhandak

    2015-01-01

    Phytol is a diterpene alcohol of medicinal importance and it also has potential to be used as biofuel. We found over production of phytol in Nostoc punctiforme by expressing a 2-Methyl-3-buten-2-ol (MBO) synthase gene. MBO synthase catalyzes the conversion of dimethylallyl pyrophosphate (DMAPP) into MBO, a volatile hemiterpene alcohol, in Pinus sabiniana. The result of enhanced phytol production in N. punctiforme, instead of MBO, could be explained by one of the 2 models: either the presence of a native prenyltransferase enzyme with a broad substrate specificity, or appropriation of a MBO synthase metabolic intermediate by a native geranyl diphosphate (GDP) synthase. In this work, an expression vector with an indigenous petE promoter for gene expression in the cyanobacterium N. punctiforme was constructed and MBO synthase gene expression was successfully shown using reverse transcriptase (RT)-PCR and SDS-PAGE. Gas chromatography – mass spectrophotometry (GC-MS) was performed to confirm phytol production from the transgenic N. punctiforme strains. We conclude that the expression of MBO synthase in N. punctiforme leads to overproduction of an economically important compound, phytol. This study provides insights about metabolic channeling of isoprenoids in cyanobacteria and also illustrates the challenges of bioengineering non-native hosts to produce economically important compounds. PMID:25424521

  3. Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

    PubMed

    Raharjo, Tri J; Chang, Wen-Te; Verberne, Marianne C; Peltenburg-Looman, Anja M G; Linthorst, Huub J M; Verpoorte, Robert

    2004-04-01

    A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

  4. Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide.

    PubMed Central

    McMillan, K; Bredt, D S; Hirsch, D J; Snyder, S H; Clark, J E; Masters, B S

    1992-01-01

    The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at approximately 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[14C]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in

  5. Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana.

    PubMed

    Kim, Won-Chan; Reca, Ida-Barbara; Kim, Yongsig; Park, Sunchung; Thomashow, Michael F; Keegstra, Kenneth; Han, Kyung-Hwan

    2014-03-01

    Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.

  6. Increased hepatic expression of nitric oxide synthase type II in cirrhotic rats

    PubMed Central

    Wang, Hai; Chen, Xiao-Ping; Qiu, Fa-Zu

    2004-01-01

    AIM: To determine the role and effect of nitric oxide synthase type II (NOS II) in cirrhotic rats. METHODS: Expression of NOS II mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO. Double-blind method was applied during the experiment. RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portal-venous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis. Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA. CONCLUSION: Increased hepatic expression of NOS II is one of the important causes of hepatic cirrhosis and portal hypertension. PMID:15222038

  7. Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

    PubMed

    Fischer, Marc J C; Meyer, Sophie; Claudel, Patricia; Perrin, Mireille; Ginglinger, Jean François; Gertz, Claude; Masson, Jean E; Werck-Reinhardt, Danièle; Hugueney, Philippe; Karst, Francis

    2013-01-10

    Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzyme's amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

  8. Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide.

    PubMed

    Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana; Nestares, Graciela

    2013-07-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first reaction in branch chain amino acids biosynthesis. This enzyme is the target of several herbicides, including all members of the imidazolinone family. Little is known about the expression of the three acetohydroxyacid synthase genes (ahas1, ahas2 and ahas3) in sunflower. The aim of this work was to evaluate ahas gene expression and AHAS activity in different tissues of sunflower plantlets. Three genotypes differing in imidazolinone resistance were evaluated, two of which carry an herbicide resistant-endowing mutation known as Ahasl1-1 allele. In vivo and in vitro AHAS activity and transcript levels were higher in leaves than in roots. The ahas3 transcript was the less abundant in both tissues. No significant difference was observed between ahas1 and ahas2 transcript levels of the susceptible genotype but a higher ahas1 transcript level was observed in leaves of genotypes carrying Ahasl1-1 allele. Similar transcript levels were found for ahas1 and ahas2 in roots of genotypes carrying Ahasl1-1 allele whereas higher ahas2 abundance was found in the susceptible genotype. Herbicide treatment triggered tissue-specific, gene and genotype-dependent changes in ahas gene expression. AHAS activity was highly inhibited in the susceptible genotype. Differential responses were observed between in vitro and in vivo AHAS inhibition assays. These findings enhance our understanding of AHAS expression in sunflower genotypes differing for herbicide resistance and its response to herbicide treatment.

  9. [Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata].

    PubMed

    Ye, Jin-cui; Zhang, Ping; Sun, Jie-yin; Guo, Chao-tan; Chen, Guo-shen; Abe, Ikuro; Noguchi, Hiroshi

    2011-10-01

    A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

  10. Structure Conservation and Differential Expression of Farnesyl Diphosphate Synthase Genes in Euphorbiaceous Plants

    PubMed Central

    Guo, Dong; Li, Hui-Liang; Peng, Shi-Qing

    2015-01-01

    Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development. PMID:26389894

  11. Pulmonary expression of nitric oxide synthase isoforms in sheep with smoke inhalation and burn injury.

    PubMed

    Cox, Robert A; Jacob, Sam; Oliveras, Gloria; Murakami, Kazunori; Enkhbaatar, Perenlei; Traber, Lillian; Schmalstieg, Frank C; Herndon, David N; Traber, Daniel L; Hawkins, Hal K

    2009-03-01

    Previous studies have indicated increased plasma levels of inducible nitric oxide synthase in lung. This study further examines the pulmonary expression of nitric oxide synthase (NOS) isoforms in an ovine model of acute lung injury induced by smoke inhalation and burn injury (S+B injury). Female range bred sheep (4 per group) were sacrificed at 4, 8, 12, 24, and 48 hours after injury and immunohistochemistry was performed in tissues for various NOS isoforms. The study indicates that in uninjured sheep lung, endothelial (eNOS) is constitutively expressed in the endothelial cells associated with the airways and parenchyma, and in macrophages. Similarly, neuronal (nNOS) is constitutively present in the mucous cells of the epithelium and in neurons of airway ganglia. In uninjured lung, inducible (iNOS) was present in bronchial secretory cells and macrophages. In tissue after S+B injury, new expression of iNOS was evident in bronchial ciliated cells, basal cells, and mucus gland cells. In the parenchyma, a slight increase in iNOS immunostaining was seen in type I cells at 12 and 24 hours after injury only. Virtually no change in eNOS or nNOS was seen after injury.

  12. Nitric oxide synthase 2 (NOS2) expression in histologically normal margins of oral squamous cell carcinoma

    PubMed Central

    Itoiz, María E.; Guiñazú, Natalia; Piccini, Daniel; Gea, Susana; López-de Blanc, Silvia

    2014-01-01

    The activity of Nitric Oxide Synthase 2 (NOS2) was found in oral squamous cell carcinomas (OSCC) but not in normal mucosa. Molecular changes associated to early carcinogenesis have been found in mucosa near carcinomas, which is considered a model to study field cancerization. The aim of the present study is to analyze NOS2 expression at the histologically normal margins of OSCC. Study Design: Eleven biopsy specimens of OSCC containing histologically normal margins (HNM) were analyzed. Ten biopsies of normal oral mucosa were used as controls. The activity of NOS2 was determined by immunohistochemistry. Salivary nitrate and nitrite as well as tobacco and alcohol consumption were also analyzed. The Chi-squared test was applied. Results: Six out of the eleven HNM from carcinoma samples showed positive NOS2 activity whereas all the control group samples yielded negative (p=0.005). No statistically significant association between enzyme expression and tobacco and/or alcohol consumption and salivary nitrate and nitrite was found. Conclusions: NOS2 expression would be an additional evidence of alterations that may occur in a state of field cancerization before the appearance of potentially malignant morphological changes. Key words:Field cancerization, oral squamous cell carcinoma, Nitric Oxide Synthase 2 (NOS2), malignity markers. PMID:24316703

  13. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase

    SciTech Connect

    Yang, Jin Won; Yoon, Se Young; Oh, Soo Jin; Kim, Sang Kyum; Kang, Keon Wook . E-mail: kwkang@chosun.ac.kr

    2006-07-21

    Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 {mu}g/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-{kappa}B (NF-{kappa}B) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects.

  14. Aminolevulinate synthase 2 mediates erythrocyte differentiation by regulating larval globin expression during Xenopus primary hematopoiesis.

    PubMed

    Ogawa-Otomo, Asako; Kurisaki, Akira; Ito, Yuzuru

    2015-01-02

    Hemoglobin synthesis by erythrocytes continues throughout a vertebrate's lifetime. The mechanism of mammalian heme synthesis has been studied for many years; aminolevulinate synthase 2 (ALAS2), a heme synthetase, is associated with X-linked dominant protoporphyria in humans. Amphibian and mammalian blood cells differ, but little is known about amphibian embryonic hemoglobin synthesis. We investigated the function of the Xenopus alas2 gene (Xalas2) in primitive amphibian erythrocytes and found that it is first expressed in primitive erythroid cells before hemoglobin alpha 3 subunit (hba3) during primary hematopoiesis and in the posterior ventral blood islands at the tailbud stage. Xalas2 is not expressed during secondary hematopoiesis in the dorsal lateral plate. Hemoglobin was barely detectable by o-dianisidine staining and hba3 transcript levels decreased in Xalas2-knockdown embryos. These results suggest that Xalas2 might be able to synthesize hemoglobin during hematopoiesis and mediate erythrocyte differentiation by regulating hba3 expression in Xenopus laevis.

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

    PubMed

    Tippmann, Stefan; Ferreira, Raphael; Siewers, Verena; Nielsen, Jens; Chen, Yun

    2017-02-09

    Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.

  16. Insect attack and wounding induce traumatic resin duct development and gene expression of (-)-pinene synthase in Sitka spruce.

    PubMed

    McKay, S Ashley Byun; Hunter, William L; Godard, Kimberley-Ann; Wang, Shawn X; Martin, Diane M; Bohlmann, Jörg; Plant, Aine L

    2003-09-01

    Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (-)-pinene synthase. The recombinant (-)-pinene synthase catalyzes the formation of (-)-alpha-pinene and (-)-beta-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (-)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.

  17. Strengthening Triterpene Saponins Biosynthesis by Over-Expression of Farnesyl Pyrophosphate Synthase Gene and RNA Interference of Cycloartenol Synthase Gene in Panax notoginseng Cells.

    PubMed

    Yang, Yan; Ge, Feng; Sun, Ying; Liu, Diqiu; Chen, Chaoyin

    2017-04-05

    To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS) was transformed into Panax notoginseng (P. notoginseng) cells in which RNA interference (RNAi) of the cycloartenol synthase (CAS) gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT) cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.

  18. Zinc affects differently growth, photosynthesis, antioxidant enzyme activities and phytochelatin synthase expression of four marine diatoms.

    PubMed

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    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.

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

    SciTech Connect

    Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano; Macone, Alberto; Boffi, Alberto; Ilari, Andrea

    2008-04-01

    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 P3{sub 1}21, 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.

  20. Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson’s disease.

    PubMed

    Abbott, Sarah K; Li, Hongyun; Muñoz, Sonia Sanz; Knoch, Bianca; Batterham, Marijka; Murphy, Karen E; Halliday, Glenda M; Garner, Brett

    2014-04-01

    Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson’s disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

  1. Genetic and epigenetic influences on expression of spermine synthase and spermine oxidase in suicide completers.

    PubMed

    Fiori, Laura M; Turecki, Gustavo

    2010-07-01

    Alterations in the levels of spermine synthase (SMS) and spermine oxidase (SMOX), two enzymes involved in polyamine metabolism, have previously been observed in brains of suicide completers. To characterize the roles played by genetic and epigenetic factors in determining expression levels of these genes, as well as to identify potential mechanisms by which to explain our findings in suicide completers, we (1) assessed the role of promoter polymorphisms in determining expression in the brain and in vitro, and (2) examined CpG methylation and levels of methylated histone H3 lysine-27 in the promoter regions of these genes in the prefrontal cortex of suicide completers and healthy controls. We identified several promoter haplotypes in SMS and SMOX, but found no consistent effects of haplotype on expression levels in either the brain or in reporter gene assays performed in three different cell lines. We also found no overall effects of epigenetic factors in determining expression, with the exception of a relationship between CpG methylation at one site in the promoter of SMOX and its expression in Brodmann area 8/9. In conclusion, the genetic and epigenetic factors examined in this study show little influence on the expression levels of SMS and SMOX, and do not appear to be responsible for the dysregulated expression of these genes in suicide completers.

  2. Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

    PubMed Central

    Wakasa, Kentaro; Kawabata, Rumi; Nakao, Seiki; Hattori, Hiroyoshi; Taguchi, Kenichi; Uchida, Junji; Yamanaka, Takeharu; Maehara, Yoshihiko; Fukushima, Masakazu; Oda, Shinya

    2015-01-01

    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. PMID:25881233

  3. Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2015-10-01

    Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.

  4. Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics

    PubMed Central

    Chen, Yunqiu; McClure, Ryan A.; Kelleher, Neil L.

    2016-01-01

    Liquid chromatography–mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products. PMID:26831706

  5. Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli.

    PubMed

    Heo, Min-Ji; Jung, Hwi-Min; Um, Jaeyong; Lee, Sang-Woo; Oh, Min-Kyu

    2017-02-17

    Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate that redistributing carbon flux using genome editing is an efficient engineering tool for metabolite overproduction.

  6. Immunohistochemical analysis of thymidylate synthase expression in gastric carcinoma: correlation with clinicopathological parameters and survival.

    PubMed

    Rogoza-Mateja, Wiesława; Domagala, Pawel; Kaczmarczyk, Mariusz; Mieżyńska-Kurtycz, Joanna; Ławniczak, Małgorzata; Sulżyc-Bielicka, Violetta; Bielicki, Dariusz; Karpińska-Kaczmarczyk, Katarzyna; Domagala, Wenancjusz

    2017-02-01

    The correlation of thymidylate synthase (TS) expression in gastric cancers with tumor histology and prognostic or predictive information remains unclear. Most studies have involved Asian populations, with few conducted in European cohorts. Moreover, all published studies analyze TS expression using semi-quantitative methods. This retrospective study evaluated the association of TS expression in tumor cells with gastric carcinoma histological type, with selected clinicopathological parameters, and with the prognosis of patients who underwent surgical treatment. TS expression was detected using immunochemistry and objectively assessed by computerized image analysis of tumor cells in 100 gastric cancers. We found that high TS expression was significantly more common in intestinal than in diffuse type of gastric cancer according to Lauren classification (P=0.0003); in type I carcinomas compared to type IV according to Goseki classification (P=0.002); and in gastric cancers in men than women (P=0.04). Low TS expression was found more often in carcinomas in the middle and lower third of the stomach than in cancers in the upper third of the stomach (P=0.009 and P=0.001, respectively). In the subgroup of 25 patients without lymph node metastases (stage I+II), high TS expression was associated with better DFS (83% for high TS expression versus 38,5% for low TS expression, P=0.03). The results (1) indicate significant correlation between the Lauren and Goseki histopathological classifications of gastric cancer and TS expression in tumor cells, (2) suggest that high TS expression may be a positive prognostic marker with regard to DFS in patients with gastric cancer without involvement of regional lymph nodes who underwent radical surgical treatment and were not treated with preoperative chemotherapy. Prognostic results need confirmation in larger cohorts.

  7. Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

    PubMed Central

    Akyürek, L. M.; Fellström, B. C.; Yan, Z. Q.; Hansson, G. K.; Funa, K.; Larsson, E.

    1996-01-01

    In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA. Images Figure 3 Figure 4 Figure 6 PMID:8952533

  8. [Cloning and expression analysis of farnesyl pyrophosphate synthase from Aquilaria sinensis].

    PubMed

    Yang, Xin; Wei, Jian-He; Liu, Juan; Xu, Yan-Hong

    2013-10-01

    Farnesyl diphosphate synthase (FPS) is one of the key rate-limiting enzymes in the sesquiterpene metabolic pathways. In this study, the open reading frame (ORF) of FPS was cloned by PCR based on the transcript sequence of AsFPS1 from the Aquilaria sinensis transcriptome database and sequenced. Total RNA was extracted from the root, stem and leaves of three-year-old A. sinensis, and from healthy and wounded A. sinensis calli, and then reverse-transcribed into single-stranded cDNA as a template for real-time PCR, to detect the expression specificity of AsFPSI in different tissues and its expression profile in responding to different treatments. The result showed that the full length of AsFPS1 was 1 342 bp with the ORF 1 029 bp, encoding 342 amino acids. Tissue expression analysis indicated that AsFPS1 was mainly expressed in root and stem, and was lower in leaves. Inducible-experiments showed that the genes was induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 12 h, respectively. The full-length cDNA clone of AsFPSI and its expression patterns analysis will provide a foundation for follow-up study on its biological function and agarwood sesquiterpene biosynthesis mechanism.

  9. Glycogen synthase kinase 3β and cyclin D1 expression in cervical carcinogenesis

    PubMed Central

    Park, Hyunsoo; Lee, Myunghwa; Kim, Dae Woon; Hong, Seo Yoo

    2016-01-01

    Objective Glycogen synthase kinase 3β (GSK3β) is a pluripotent protein kinase involved in the development of cancers through regulation of numerous oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of target proteins that GSK3β regulate. Our objective was to assess the expression of GSK3β and cyclin D1 in cervical neoplasm of different histologic grades and to identify their correlation in cervical carcinogenesis. Methods Immunohistochemical analysis of GSK3β and cyclin D1 was performed in a total of 137 patients with 12 normal, 62 cervical intraepithelial neoplasia (CIN) (31 CIN1 and 31 CIN3) and 63 invasive cancers including 56 squamous cell carcinomas and 7 adenocarcinomas. Results The expression of GSK3β increased in parallel with the lesion grade, while that of cyclin D1 decreased with severity of the lesion (P<0.001). There was a significant inverse correlation between GSK3β and cyclin D1 expression in overall cervical neoplasia (Φ=-0.413, P<0.001). GSK3β expression was higher in squamous cell carcinoma than in adenocarcinoma (P=0.049). Conclusion These results suggest that the expressional increase in GSK3β plays a role in cervical carcinogenesis and has inverse correlation with cyclin D1 expression in this process. In addition, GSK3β expression appears to be associated with the histologic type of cervical cancer, especially squamous cell carcinoma. PMID:27896249

  10. Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.

    PubMed

    Sierra, Ana; Navascués, Julio; Cuadros, Miguel A; Calvente, Ruth; Martín-Oliva, David; Ferrer-Martín, Rosa M; Martín-Estebané, María; Carrasco, María-Carmen; Marín-Teva, José L

    2014-01-01

    Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid

  11. Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

    PubMed

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

    2010-07-01

    Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta

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

    SciTech Connect

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. )

    1991-06-01

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

  13. The human ATP synthase beta subunit gene: sequence analysis, chromosome assignment, and differential expression.

    PubMed

    Neckelmann, N; Warner, C K; Chung, A; Kudoh, J; Minoshima, S; Fukuyama, R; Maekawa, M; Shimizu, Y; Shimizu, N; Liu, J D

    1989-11-01

    In humans, the functional F0F1-ATP synthase beta subunit gene is located on chromosome 12 in the p13----qter region. Other partially homologous sequences have been detected on chromosomes 2 and 17. The bona fide beta subunit gene has 10 exons encoding a leader peptide of 49 amino acids and a mature protein of 480 amino acids. Thirteen Alu family DNA repeats are found upstream from the gene and in four introns. The gene has four "CCAAT" sequences upstream and in close proximity to the transcriptional initiation site. A 13-bp motif is found in the 5' nontranscribed region of both the beta subunit gene and an ADP/ATP translocator gene that is expressed in high levels in cardiac and skeletal muscle. Analysis of the beta subunit mRNA levels reveals marked differences among tissues. The highest levels are found in heart, lower levels in skeletal muscle, and the lowest levels in liver and kidney. These findings suggest that the tissue-specific levels of ATP synthase beta subunit mRNA may be generated through transcriptional control.

  14. Expression of parsley flavone synthase I establishes the flavone biosynthetic pathway in Arabidopsis thaliana.

    PubMed

    Yun, Choong-Soo; Yamamoto, Tomio; Nozawa, Akira; Tozawa, Yuzuru

    2008-04-01

    Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.

  15. The phytochelatin synthase gene in date palm (Phoenix dactylifera L.): Phylogeny, evolution and expression.

    PubMed

    Zayneb, Chaâbene; Imen, Rekik Hakim; Walid, Kriaa; Grubb, C Douglas; Bassem, Khemakhem; Franck, Vandenbulcke; Hafedh, Mejdoub; Amine, Elleuch

    2017-06-01

    We studied date palm phytochelatin synthase type I (PdPCS1), which catalyzes the cytosolic synthesis of phytochelatins (PCs), a heavy metal binding protein, in plant cells. The gene encoding PdPCS1 (Pdpcs) consists of 8 exons and 7 introns and encodes a protein of 528 amino acids. PCs gene history was studied using Notung phylogeny. During evolution, gene loss from several lineages was predicted including Proteobacteria, Bilateria and Brassicaceae. In addition, eleven gene duplication events appeared toward interior nodes of the reconciled tree and four gene duplication events appeared toward the external nodes. These latter sequences belong to species with a second copy of PCs suggesting that this gene evolved through subfunctionalization. Pdpcs1 gene expression was measured in seedling hypocotyls exposed to Cd, Cu and Cr using quantitative real-time polymerase chain reaction (qPCR). A Pdpcs1 overexpression was evidenced in P. dactylifera seedlings exposed to metals suggesting that 1-the Pdpcs1 gene is functional, 2-there is an implication of the enzyme in metal detoxification mechanisms. Additionally, the structure of PdPCS1 was predicted using its homologue from Nostoc (cyanobacterium, NsPCS) as a template in Discovery studio and PyMol software. These analyses allowed us to identify the phytochelatin synthase type I enzyme in date palm (PdPCS1) via recognition of key consensus amino acids involved in the catalytic mechanism, and to propose a hypothetical binding and catalytic site for an additional substrate binding cavity.

  16. Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise.

    PubMed

    Shen, W; Zhang, X; Zhao, G; Wolin, M S; Sessa, W; Hintze, T H

    1995-08-01

    Nitric oxide (NO) is a vasodilator produced under normal physiologic conditions primarily by the vascular endothelium lining all blood vessels. The primary stimulus for the production of nitric oxide by the constitutive endothelial nitric oxide synthase (ECNOS, Type II) found in blood vessels is most likely the shear stress, the frictional force, caused by blood flowing through blood vessels. During exercise there is an increase in cardiac output and redistribution of blood flow to increase blood flow in skeletal muscle and in the coronary circulation. These adjustments provide increased oxygen delivery to support aerobic energy production and to sustain the exercise response. NO may be involved in the regulation of vascular tone in exercising skeletal and cardiac muscle by promoting, enhancing the metabolic vasodilation. In addition, the production of NO by capillary endothelium may regulate oxygen consumption by mitochondria through chemical interactions between NO and the iron-sulfur center of these enzymes. Finally, brief exercise training may alter the gene expression for the enzyme, the constitutive endothelial NO synthase, which forms NO and may be part of the vascular adaptation seen after aerobic exercise training. Furthermore, if there is a genetic predisposition to produce NO, as in world class athletes or animals bred to race, NO may contribute to spectacular exercise performance. These three potential roles of NO will be discussed and data presented to support each of these in our review.

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

    PubMed

    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.

  18. Expression and regulation of glycogen synthase kinase 3 in human neutrophils.

    PubMed

    Giambelluca, Miriam S; Cloutier, Nathalie; Rollet-Labelle, Emmanuelle; Boilard, Eric; Pouliot, Marc

    2013-11-01

    Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in the regulation of cellular processes ranging from glycogen metabolism to cell cycle regulation. Its two known isoforms, α and β, are differentially expressed in tissues throughout the body and exert distinct but often overlapping functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3α) or Ser9 (GSK-3β) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system function, yet little is known about the role of this enzyme in neutrophils, the most abundant leukocyte type. In the present study, we examined GSK-3 expression and regulation in human neutrophils. GSK-3α was found to be the predominant isoform, it was constitutively expressed and cell stimulation with different agonists did not alter its expression. Stimulation by fMLP, LPS, GM-CSF, Fcγ receptor engagement, or adenosine A2A receptor engagement all resulted in phosphorylation of Ser21. The use of metabolic inhibitors revealed that combinations of Src kinase, PKC, PI3K/AKT, ERK/RSK and PKA signaling pathways could mediate phosphorylation, depending on the agonist. Neither PLC nor p38 were involved. We conclude that GSK-3α is the main isoform expressed in neutrophils and that many different pathways can converge to inhibit GSK-3α activity via Ser21-phosphorylation. GSK-3α thus might be a hub of cellular regulation.

  19. Expression and tissue and subcellular localization of anthocyanidin synthase (ANS) in grapevine.

    PubMed

    Wang, Huiling; Wang, Wei; Li, Hui; Zhang, Ping; Zhan, Jicheng; Huang, Weidong

    2011-04-01

    Anthocyanidin synthase (ANS) is one of the key enzymes in the biosynthesis of both anthocyanins and proanthocyanidins in grapevine. Although substantial researches have investigated ANS gene expression and regulation at the transcriptional level, little is yet known about protein expression and distribution in grapevine. Here, the expression and tissue and subcellular localization of ANS in different Cabernet sauvignon grapevine tissues were investigated by using the techniques of Western blotting, immunohistochemical localization, immuno-electron microscopy, and confocal microscopy. The results showed that the ANS was expressed in the grape berries, leaves, stems, petioles, and leaf buds. In grape berry skin and flesh, ANS expression is developmental dependent. Immunohistochemical analysis revealed that ANS is primarily distributed in the exocarp, mesocarp, and seed of the fruit; in palisade and spongy tissues of the leaves; in the primary phloem and pith ray in the stems; and in the growth point and leaf primordium of the leaf buds. Furthermore, at the subcellular level, the ANS was mainly localized in the cytoplasm regardless of cell types and some ANS were also found in the nucleus in the mesocarp vascular bundle and leaf bud cells. This research will give further insight for the biosynthesis and regulation of different flavonoid compounds in grapevine.

  20. Cloning and analysis of valerophenone synthase gene expressed specifically in lupulin gland of hop (Humulus lupulus L.).

    PubMed

    Okada, Y; Ito, K

    2001-01-01

    Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of alpha-acid and beta-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland.

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

    PubMed

    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.

  2. Cloning and expression of trehalose-6-phosphate synthase 1 from Rhizopus oryzae.

    PubMed

    Ozer Uyar, Ebru; Yücel, Meral; Hamamcı, Haluk

    2016-05-01

    Trehalose is a reducing disaccharide acting as a protectant against environmental stresses in many organisms. In fungi, Trehalose-6-phosphate synthase 1 (TPS1) plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Rhizopus oryzae encoding TPS1 (designated as RoTPS1) was isolated. The RoTPS1 cDNA is composed of 2505 nucleotides and encodes a protein of 834 amino acids with a molecular mass of 97.8 kDa. The amino acid sequence of RoTPS1 has a relatively high homology with the TPS1s in several other filamentous fungi. RoTPS1 was cloned into Saccharomyces cerevisiae and secretively expressed.

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

    PubMed

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

    2013-01-01

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

  4. Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

    PubMed Central

    Nordström, Viola; Willershäuser, Monja; Herzer, Silke; Rozman, Jan; von Bohlen und Halbach, Oliver; Meldner, Sascha; Rothermel, Ulrike; Kaden, Sylvia; Roth, Fabian C.; Waldeck, Clemens; Gretz, Norbert; de Angelis, Martin Hrabě; Draguhn, Andreas; Klingenspor, Martin

    2013-01-01

    Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis. PMID:23554574

  5. Apoptotic effect of tannic acid on fatty acid synthase over-expressed human breast cancer cells.

    PubMed

    Nie, Fangyuan; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-02-01

    Breast cancer is one of the most common cancers and is the second leading cause of cancer mortality in women worldwide. Novel therapies and chemo-therapeutic drugs are urgently needed to be developed for the treatment of breast cancer. Increasing evidence suggests that fatty acid synthase (FAS) plays an important role in breast cancer, for the expression of FAS is significantly higher in human breast cancer cells than in normal cells. Tannic acid (TA), a natural polyphenol, possesses significant biological functions, including bacteriostasis, hemostasis, and anti-oxidant. Our previous studies demonstrated that TA is a natural FAS inhibitor whose inhibitory activity is stronger than that of classical FAS inhibitors, such as C75 and cerulenin. This study further assessed the effect and therapeutic potential of TA on FAS over-expressed breast cancer cells, and as a result, TA had been proven to possess the functions of inhibiting intracellular FAS activity, down-regulating FAS expression in human breast cancer MDA-MB-231 and MCF-7 cells, and inducing cancer cell apoptosis. Since high-expressed FAS is recognized as a molecular marker for breast cancer and plays an important role in cancer prognosis, these findings suggest that TA is a potential drug candidate for treatment of breast cancer.

  6. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    PubMed

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  7. Expression of Monoamine Transporters, Nitric Oxide Synthase 3, and Neurotrophin Genes in Antidepressant-Stimulated Astrocytes

    PubMed Central

    Kittel-Schneider, Sarah; Kenis, Gunter; Schek, Julia; van den Hove, Daniel; Prickaerts, Jos; Lesch, Klaus-Peter; Steinbusch, Harry; Reif, Andreas

    2012-01-01

    Background: There is increasing evidence that glial cells play a role in the pathomechanisms of mood disorders and the mode of action of antidepressant drugs. Methods: To examine whether there is a direct effect on the expression of different genes encoding proteins that have been implicated in the pathophysiology of affective disorders, primary astrocyte cell cultures from rats were treated with two different antidepressant drugs, imipramine and escitalopram, and the RNA expression of brain-derived neurotrophic factor (Bdnf), serotonin transporter (5Htt), dopamine transporter (Dat), and endothelial nitric oxide synthase (Nos3) was examined. Results: Stimulation of astroglial cell culture with imipramine, a tricyclic antidepressant, led to a significant increase of the Bdnf RNA level whereas treatment with escitalopram did not. In contrast, 5Htt was not differentially expressed after antidepressant treatment. Finally, neither Dat nor Nos3 RNA expression was detected in cultured astrocytes. Conclusion: These data provide further evidence for a role of astroglial cells in the molecular mechanisms of action of antidepressants. PMID:22529824

  8. Molecular cloning and expression pattern of oriental river prawn (Macrobrachium nipponense) nitric oxide synthase.

    PubMed

    Rahman, N M A; Fu, H T; Sun, S M; Qiao, H; Jin, S; Bai, H K; Zhang, W Y; Liang, G X; Gong, Y S; Xiong, Y W; Wu, Y

    2016-08-29

    Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of l-arginine to l-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide phosphate. Recently, various studies have verified the importance of NOS invertebrates and invertebrates. However, the NOS gene family in the oriental river prawn Macrobrachium nipponense is poorly understood. In this study, we cloned the full-length NOS complementary DNA from M. nipponense (MnNOS) and characterized its expression pattern in different tissues and at different developmental stages. Real-time quantitative polymerase chain reaction (RT-qPCR) showed the MnNOS gene to be expressed in all investigated tissues, with the highest levels observed in the androgenic gland (P < 0.05). Our results revealed that the MnNOS gene may play a key role in M. nipponense male sexual differentiation. Moreover, RT-qPCR revealed that MnNOS mRNA expression was significantly increased in post-larvae 10 days after metamorphosis (P < 0.05). The expression of this gene in various tissues indicates that it may perform versatile biological functions in M. nipponense.

  9. 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

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

    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.

  10. 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

    DOEpatents

    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.

  11. Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines.

    PubMed

    Niranjan, S K; Goyal, S; Dubey, P K; Kumari, N; Mishra, S K; Mukesh, M; Kataria, R S

    2016-01-10

    Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines

  12. Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

    PubMed

    Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

    2001-10-01

    Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These

  13. Altered Composition of Ralstonia eutropha Poly(hydroxyalkanoate) through Expression of PHA Synthase from Allochromatium vinosum ATCC 35206

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. We employed a PCR technique coupled with a chromosomal gene-walking method to clone and subsequen...

  14. Primary aldosteronism and pregnancy.

    PubMed

    Landau, Ester; Amar, Laurence

    2016-06-01

    Hypertension (HT) is a complication of 8% of all pregnancies and 10% of HT cases are due to primary aldosteronism (PA). There is very little data on PA and pregnancy. Given the changes in the renin angiotensin system during pregnancy, the diagnosis of PA is difficult to establish during gestation. It may be suspected in hypertensive patients with hypokalemia. A comprehensive literature review identified reports covering 40 pregnancies in patients suffering from PA. Analysis of these cases shows them to be high-risk pregnancies leading to maternal and fetal complications. Pregnancy must be programmed, and if the patient has a unilateral form of PA, adrenalectomy should be performed prior to conception. It is customary to stop spironolactone prior to conception and introduce antihypertensive drugs that present no risk of teratogenicity. When conventional antihypertensive drugs used during pregnancy fail to control high blood pressure, diuretics, including potassium-sparing diuretics may be prescribed. Adrenalectomy can be considered during the second trimester of pregnancy exclusively in cases of refractory hypertension. A European retrospective study is currently underway to collect a larger number of cases.

  15. Expression patterns, activities and carbohydrate-metabolizing regulation of sucrose phosphate synthase, sucrose synthase and neutral invertase in pineapple fruit during development and ripening.

    PubMed

    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.

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

    PubMed Central

    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

  17. Hypergravity upregulates renal inducible nitric oxide synthase expression and nitric oxide production

    PubMed Central

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-01-01

    Exposure to hypergravity severely decreases renal blood flow, potentially causing renal dysfunction. Nitric oxide (NO), which is endogenously synthesized by inducible NO synthase (iNOS), plays an important role in the regulation of renal function. The purpose of this study was to examine the effect of hypergravity exposure on the production of NO in kidneys. To determine whether hypergravity induces renal hypoxia and alters renal iNOS expression and NO production, mice were exposed to short-term hypergravity at +3Gz for 1 h. The time course of iNOS mRNA expression, hypoxia-inducible factor (HIF)-1α expression, and NO production was examined. Renal HIF-1α levels were significantly elevated immediately after centrifugation, and this increase was sustained for 3 h post-exposure. iNOS mRNA levels were also significantly increased immediately after exposure and were maintained during the reoxygenation period. Immunohistochemical staining for iNOS revealed that the cortical tubular epithelium exhibited moderate to strong cytoplasmic iNOS immunoreactivity immediately after hypergravity exposure and during the reoxygenation period. The time course of NO production was similar to that of iNOS expression. Our results suggest that both hypoxia and reoxygenation might be involved in the upregulation of HIF-1α in the kidneys of mice exposed to hypergravity. Significant increases in renocortical iNOS expression immediately after centrifugation and during the reoxygenation period suggest that iNOS expression induced by hypergravity exposure might play a protective role against hypoxia/reoxygenation injury in the renal cortex. Further investigations are necessary to clarify the role of iNOS and NO in kidneys exposed to hypergravity. PMID:27174912

  18. Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta.

    PubMed

    Förstermann, U; Schmidt, H H; Kohlhaas, K L; Murad, F

    1992-02-13

    RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.

  19. Influence of Different Levels of Lipoic Acid Synthase Gene Expression on Diabetic Nephropathy

    PubMed Central

    Xu, Longquan; Hiller, Sylvia; Simington, Stephen; Nickeleit, Volker; Maeda, Nobuyo; James, Leighton R.; Yi, Xianwen

    2016-01-01

    Oxidative stress is implicated in the pathogenesis of diabetic nephropathy (DN) but outcomes of many clinical trials are controversial. To define the role of antioxidants in kidney protection during the development of diabetic nephropathy, we have generated a novel genetic antioxidant mouse model with over- or under-expression of lipoic acid synthase gene (Lias). These models have been mated with Ins2Akita/+ mice, a type I diabetic mouse model. We compare the major pathologic changes and oxidative stress status in two new strains of the mice with controls. Our results show that Ins2Akita/+ mice with under-expressed Lias gene, exhibit higher oxidative stress and more severe DN features (albuminuria, glomerular basement membrane thickening and mesangial matrix expansion). In contrast, Ins2Akita/+ mice with highly-expressed Lias gene display lower oxidative stress and less DN pathologic changes. Our study demonstrates that strengthening endogenous antioxidant capacity could be an effective strategy for prevention and treatment of DN. PMID:27706190

  20. Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae.

    PubMed

    Jung, Won Seok; Lee, Sang Kil; Hong, Jay Sung Joong; Park, Sung Ryeol; Jeong, Soon Jeong; Han, Ah Reum; Sohng, Jae Kyung; Kim, Byung Gee; Choi, Cha Yong; Sherman, David H; Yoon, Yeo Joon

    2006-10-01

    Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.

  1. Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis.

    PubMed

    Cai, Quanxiang; Zhao, Mengmeng; Liu, Xiang; Wang, Xiaochun; Nie, Yao; Li, Ping; Liu, Tingyan; Ge, Ruli; Han, Fengchan

    2017-02-16

    A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

  2. Repeated thermal therapy upregulates arterial endothelial nitric oxide synthase expression in Syrian golden hamsters.

    PubMed

    Ikeda, Y; Biro, S; Kamogawa, Y; Yoshifuku, S; Eto, H; Orihara, K; Kihara, T; Tei, C

    2001-05-01

    It has been previously reported that sauna therapy, a thermal therapy, improves the hemodynamics and clinical symptoms in patients with chronic heart failure and also improves endothelial function, which is impaired in such patients. The present study investigated whether the improvements observed with sauna therapy are through modulation of arterial endothelial nitric oxide synthase (eNOS) expression. Eight male Syrian golden hamsters underwent sauna therapy, using an experimental far infrared-ray dry sauna system, at 39 degrees C for 15 min followed by 30 degrees C for 20 min daily for 4 weeks. Control group hamsters were placed in the sauna system switched off at room temperature of 24 degrees C for 35 min. Immunohistochemistry found greater amounts of the immunoreactive products of eNOS in the endothelial cells of the aorta and carotid, femoral and coronary arteries in the sauna group than in the control group. Western blot analysis also revealed that 4-week sauna therapy significantly increased eNOS expression in aortas by 50% in 4 series of independent experiments with an identical protocol (p<0.01). In reverse transcription polymerase chain reaction assay, the eNOS mRNA in aortas was greater in the sauna group than in controls, with a peak at 1-week of sauna therapy (approximately 40-fold increase). In conclusion, repeated thermal therapy upregulates eNOS expression in arterial endothelium.

  3. Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene.

    PubMed Central

    Xu, W; Warner, C A; Desnick, R J

    1995-01-01

    To investigate the molecular basis of the phenotypic heterogeneity in congenital erythropoietic porphyria, the mutations in the uroporphyrinogen III synthase gene from unrelated patients were determined. Six missense (L4F, Y19C, V82F, V99A, A104V, and G225S), a nonsense (Q249X), a frameshift (633insA), and two splicing mutations (IVS2+1 and IVS9 delta A + 4) were identified. When L4F, Y19C, V82F, V99A, A104V, 633insA, G225S, and Q249X were expressed in Escherichia coli, only the V82F, V99A, and A104V alleles expressed residual enzymatic activity. Of note, the V82F mutation, which occurs adjacent to the 5' donor site of intron 4, resulted in approximately 54% aberrantly spliced transcripts with exon 4 deleted. Thus, this novel exonic single-base substitution caused two lesions, a missense mutation and an aberrantly spliced transcript. Of the splicing mutations, the IVS2+1 allele produced a single transcript with exon 2 deleted, whereas the IVS9 delta A+4 allele was alternatively spliced, approximately 26% being normal transcripts and the remainder with exon 9 deleted. The amount of residual activity expressed by each allele provided a basis to correlate genotype with disease severity, thereby permitting genotype/phenotype predictions in this clinically heterogeneous disease. Images PMID:7860775

  4. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients.

    PubMed

    St Clair, E W; Wilkinson, W E; Lang, T; Sanders, L; Misukonis, M A; Gilkeson, G S; Pisetsky, D S; Granger, D I; Weinberg, J B

    1996-09-01

    Nitric oxide (NO) is an important inflammatory mediator in nonhuman animal models of rheumatoid arthritis (RA). The purpose of the present study was to determine whether blood mononuclear cells from patients with active RA (as compared to control subjects) have higher levels of NO synthase type 2 (NOS2) and produce more NO in vitro. Leukocytes from 25 RA patients and 20 normal subjects were examined. Arthritis activity was assessed by tender and swollen joint counts, duration of morning stiffness, patient assessment of pain, physician and patient global assessment of disease activity, the modified Stanford Health Assessment Questionnaire, and by blood levels of acute phase reactants. Blood mononuclear cell NOS enzyme activity/antigen content and nitrite/nitrate formation in vitro were measured. Blood mononuclear cells from RA patients had increased NOS activity and increased NOS2 antigen content as compared to those from normal subjects, and responded to interferon-gamma with increased NOS expression and nitrite/nitrate production in vitro. NOS activity of freshly isolated blood mononuclear cells correlated significantly with disease activity, as assessed by render and swollen joint counts. Our results demonstrate that patients with RA have systemic activation for NOS2 expression, and that the degree of activation correlates with disease activity. Increased NOS2 expression and NO generation may be important in the pathogenesis of RA.

  5. Aldosterone and the vascular system.

    PubMed

    Cachofeiro, Victoria; Miana, Maria; de Las Heras, Natalia; Martín-Fernández, Beatriz; Ballesteros, Sandra; Fernández-Tresguerres, Jesús; Lahera, Vicente

    2008-04-01

    Aldosterone can act in different tissues exerting physiological and pathological effects. At the vascular level, aldosterone affects endothelial function since administration of aldosterone impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorate relaxation to acetylcholine in models of both hypertension and atherosclerosis and in patients with heart failure. A reduction in nitric oxide levels seems to be the main mechanism underlying this effect due to a reduction in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone is a pro-inflammatory factor that can participate in the vascular inflammatory process associated with different pathologies including hypertension through activation of the NFkappaB system, which mediates the vascular production of different cytokines. This mineralocorticoid also participates in the vascular remodeling observed in hypertensive rats since the administration of eplerenone improved the media-to-lumen ratio in these animals. This effect seems to be due to an increase in extracellular matrix. In summary, aldosterone through mineralocorticoid receptors can participate in the vascular damage associated with different pathologies including hypertension through its prooxidant, pro-inflammatory and profibrotic effects that triggered endothelial dysfunction, an inflammatory process and vascular remodeling.

  6. SY 03-3 OVERVIEW OF SOMATIC MUTATIONS AND EPIGENETIC REGULATION OF ALDOSTERONE PRODUCING ADENOMA (APA).

    PubMed

    Umemura, Satoshi

    2016-09-01

    clear cells with abundant microvesicular cytoplasm with numerous lipid droplets ('fasciculata-like'). The histological pattern of tumors with CACNA1D and ATPase mutations was still controversial (Clin Endo 2015; 83: 779, JCEM 2016;101:494)(3) Epigenetics in APAIntegrated analysis of genome-wide methylation and gene expression shows epigenetic regulation of CYP11B2 in aldosteronomas. APA were hypomethylated compared with normal, nonfunctioning adrenocortical tumors and APA-adjacent adrenal gland samples from the same patient. CYP11B2 was upregulated and hypomethylated, suggesting that dysregulated methylation preferentially upregulates CYP11B2 with commensurate inhibition of other key genes involved in steroid biosynthesis. Comparing the methylation status of CYP11B2 CpG islands in paired DNA samples from aldosteronomas and blood from the same patients showed CpG hypomethylation was found in aldosteronomas but not in peripheral blood DNA in all cases. These results may suggest the involvement of these DNA hypomethylation in aldosterone production. (J Clin Endocrinol Metab 2014; 99: E536. Eur J Endocrinol 2015; 173: 185)(4) Aldosterone-producing cell clusters (APCCs) with high expression of CYP11B2 in both normal and PA adrenal tissueRecently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. Known aldosterone driver mutations were identified in some of APCCs, including CACNA1D and ATP1A1, which were not observed in the adjacent normal adrenal tissue. These studies suggest that APCCs are common in normal adrenals, and APCCs harbor somatic mutations known to cause excess aldosterone production. Furthermore, the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. The mutations in KCNJ5 were not detected in those APCCs. The frequency of somatic mutations in CACNA1D in APCCs from normal adrenals increases with age. These results may

  7. Inhibition of inducible nitric oxide synthase expression by yuccaol C from Yucca schidigera roezl.

    PubMed

    Marzocco, Stefania; Piacente, Sonia; Pizza, Cosimo; Oleszek, Wieslaw; Stochmal, Anna; Pinto, Aldo; Sorrentino, Raffaella; Autore, Giuseppina

    2004-08-06

    Yucca schidigera extract finds wide commercial application in foods, cosmetics and pharmaceuticals. In a previous paper we have found as the main constituents of yucca bark, yuccaol A, B and C, new and very unusual spiro-derivatives made up of a C15 unit and a stilbenic portion closely related to resveratrol. This study was performed to examine whether yuccaol A, B or C (0.01-100 microM) could affect cytosolic inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) generation in vitro in Escherichia coli lipopolysaccharide (LPS)-activated J774.A1 macrophage cell line. NO production, detected as NO2-, increased significantly in LPS treated J774.A1 cells from 0.05 +/- 0.03 microM to 16.64 +/- 0.58 microM (P < 0.001). Yuccaol C (0.01-100 microM), added to the culture medium 1 h before LPS-stimulation, significantly (P < 0.001) and in a concentration related manner reduced NO release (P < 0.001) and iNOS protein expression (P < 0.05). In contrast, no inhibitory effect either on iNOS protein expression or on NO release was observed when yuccaol C was added after LPS stimulation. In contrast yuccaol A inhibited significantly (P < 0.001) only NO release at the highest concentration tested (100 microM) while yuccaol B did not reduce either NO release or iNOS expression. Yuccaol C was demonstrated to reduce iNOS protein expression via the transcription factor NF-kappaB. These results indicated that the empirical use of Y. schidigera as anti-inflammatory remedy could be addressed not only to the resveratrol content but also to the presence of yuccaol C.

  8. Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

    PubMed

    Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

    2016-01-01

    Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

  9. Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine aortic endothelial cells

    NASA Technical Reports Server (NTRS)

    Segurola, R. J. Jr; Oluwole, B.; Mills, I.; Yokoyama, C.; Tanabe, T.; Kito, H.; Nakajima, N.; Sumpio, B. E.

    1997-01-01

    Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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<0.05) than in leaves. Lead and mercury exposure significantly enhanced the mRNA expression of SsPCS ( P<0.05). To the best of our knowledge, SsPCS is the second PCS gene cloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  11. Effects of phenylethanoid glycosides from Digitalis purpurea L. on the expression of inducible nitric oxide synthase.

    PubMed

    Oh, Jae Wook; Lee, Jeong Yong; Han, Song Hee; Moon, Young Hee; Kim, Yoon Gyoon; Woo, Eun-Rhan; Kang, Keon Wook

    2005-07-01

    We have isolated four different phenylethanoid glycosides (purpureaside A, desrhamnosyl acteoside, calceolarioside B and plantainoside D) from the leaves of Digitalis purpurea (foxglove). The effects of these glycosides on activator protein-1 (AP-1)-mediated inducible nitric oxide synthase (iNOS) gene expression in the Raw264.7 macrophage cell line have been studied. Of these four glycosides, purpureaside A potently inhibited iNOS induction by lipopolysaccharide (LPS). Increase in iNOS mRNA by LPS was completely suppressed by purpureaside A. Purpureaside A did not significantly affect LPS-inducible nuclear factor-kappaB (NF-kappaB) activation or the nuclear translocation of p65. Moreover, a reporter gene assay using AP-1 specific luciferase reporter revealed that the enhanced activity of AP-1 by LPS was completely abolished in cells treated with purpureaside A. These results demonstrated that purpureaside A inhibited LPS-inducible iNOS expression in macrophages through the suppression of AP-1, but not of NF-kappaB.

  12. Constitutive expression of mammalian nitric oxide synthase in tobacco plants triggers disease resistance to pathogens.

    PubMed

    Chun, Hyun Jin; Park, Hyeong Cheol; Koo, Sung Cheol; Lee, Ju Huck; Park, Chan Young; Choi, Man Soo; Kang, Chang Ho; Baek, Dongwon; Cheong, Yong Hwa; Yun, Dae-Jin; Chung, Woo Sik; Cho, Moo Je; Kim, Min Chul

    2012-11-01

    Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H(2)O(2), with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.

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

    PubMed Central

    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

  14. Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants.

    PubMed

    Song, Jian; Liu, Jing; Weng, Manli; Huang, Yanyan; Luo, Lei; Cao, Pengxiu; Sun, Haiwei; Liu, Jie; Zhao, Jinhong; Feng, Dianqi; Wang, Bin

    2013-01-15

    A cold induced galactinol synthase gene (AmGS) and its promoter sequence were identified and cloned from the cold-tolerant tree Ammopiptanthus mongolicus by using cDNA-AFLP, RACE-PCR and TAIL-PCR strategies combined with its expression pattern analysis after cold inducing treatment. Accession number of the AmGS gene in GenBank is DQ519361. The open reading frame (ORF) region of the AmGS gene is 987 nucleotides encoding for 328 amino acid residues and a stop codon. The genomic DNA sequence of AmGS gene contains 3 exons and 2 introns. Moreover, a variety of temporal gene expression patterns of AmGS was detected, which revealed the up-regulation of AmGS gene in stresses of cold, ABA and others. Then the AmGS gene was transformed into Photinia serrulata tree by Agrobacterium-mediated transformation, and the transgenic plants exhibited higher cold-tolerance comparing with non-transformed plants.

  15. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

    PubMed

    Yang, Jeong-Yeh; Koo, Bon-Sun; Kang, Mi-Kyung; Rho, Hye-Won; Sohn, Hee-Sook; Jhee, Eun-Chung; Park, Jin-Woo

    2002-11-30

    The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.

  16. Nicotinamide increases thyroid radiosensitivity by stimulating nitric oxide synthase expression and the generation of organic peroxides.

    PubMed

    Agote Robertson, M; Finochietto, P; Gamba, C A; Dagrosa, M A; Viaggi, M E; Franco, M C; Poderoso, J J; Juvenal, G J; Pisarev, M A

    2006-01-01

    Differentiated thyroid cancer and hyperthyroidism are treated with radioiodine. However, when the radioisotope dose exceeds certain limits, the patient must be hospitalized to avoid contact with people that would otherwise be exposed to radiation. It would be desirable to obtain a similar therapeutic effect using lower radioiodine doses. Radiosensitizers can be utilized for this purpose. Nicotinamide (NA) increases thyroid radiosensitivity to 131I in both normal and goitrous glands. NA causes a significant increase in thyroid blood flow, which would increase tissue oxygenation and tissue damage via free radicals. Wistar rats were treated with either nicotinamide (NA), 131I or both. The expression of the three isoforms of nitric oxide synthase (NOS) in the thyroid (Western blot) and the activities of SOD, GPx, catalase and organic peroxides were determined. Treatment with NA or 131I increased the expression of eNOS and the generation of organic peroxides. When administered jointly, they showed a synergistic effect. No changes were observed in the other NOS isoforms or in the activities of catalase, glutathione peroxidase and superoxide dismutase. NA potentiates the effect of 131I by increasing eNOS, which would in turn stimulate NO production, increasing thyroid blood flow and tissue damage via organic peroxides.

  17. TASK channels are not required to mount an aldosterone secretory response to metabolic acidosis in mice

    PubMed Central

    Guagliardo, Nick A.; Yao, Junlan; Bayliss, Douglas A.; Barrett, Paula Q.

    2010-01-01

    The stimulation of aldosterone production by acidosis enhances proton excretion and serves to limit disturbances in systemic acid-base equilibrium. Yet, the mechanisms by which protons stimulate aldosterone production from cells of the adrenal cortex remain largely unknown. TWIK-related acid sensitive K channels (TASK) are inhibited by extracellular protons within the physiological range and have emerged as important regulators of aldosterone production in the adrenal cortex. Here we show that congenic C57BL/6J mice with genetic deletion of TASK-1 (K2P3.1) and TASK-3 (K2P9.1) channel subunits overproduce aldosterone and display an enhanced sensitivity to steroidogenic stimuli, including a more pronounced steroidogenic response to chronic NH4Cl loading. Thus, we conclude that TASK channels are not required for the stimulation of aldosterone production by protons but their inhibition by physiological acidosis may contribute to full expression of the steroidogenic response. PMID:21111026

  18. Genome Mining in Streptomyces clavuligerus. Expression and Biochemical Characterization of Two Cryptic Sesquiterpene Cyclases, (−)-δ-Cadinene Synthase and (+)-T-Muurolol Synthase

    PubMed Central

    Hu, Yunfeng; Chou, Wayne K. W.; Hopson, Russell; Cane, David E.

    2010-01-01

    SUMMARY Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His6-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (−)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (−)-δ-cadinene synthase with [1,1-2H2]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (−)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1si of FPP, cyclization and deprotonation will give (−)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1siof FPP. PMID:21276937

  19. E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

    PubMed

    Lawrence, Donald W; Gullickson, Gail; Kornbluth, Jacki

    2015-01-01

    Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated NKLAM-KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNβ). The JAK/STAT pathway is critical for the production of IFNβ, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

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

    SciTech Connect

    Yoshigai, Emi; Machida, Toru; Okuyama, Tetsuya; Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota; Okumura, Tadayoshi; Ikeya, Yukinobu; Nishino, Hoyoku; Nishizawa, Mikio

    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.

  1. Characterization and Expression Analysis of Phytoene Synthase from Bread Wheat (Triticum aestivum L.)

    PubMed Central

    Flowerika; Alok, Anshu; Kumar, Jitesh; Thakur, Neha; Pandey, Ashutosh; Pandey, Ajay Kumar; Upadhyay, Santosh Kumar; Tiwari, Siddharth

    2016-01-01

    Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop. PMID:27695116

  2. Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae.

    PubMed

    Valenzuela, L; Ballario, P; Aranda, C; Filetici, P; González, A

    1998-07-01

    Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.

  3. Cloning, Expression, and Characterization of cis-Polyprenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius

    PubMed Central

    Hemmi, Hisashi; Yamashita, Satoshi; Shimoyama, Takefumi; Nakayama, Toru; Nishino, Tokuzo

    2001-01-01

    cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme. PMID:11114943

  4. Expression Pattern and Biochemical Properties of Zebrafish N-Acetylglutamate Synthase

    PubMed Central

    Caldovic, Ljubica; Haskins, Nantaporn; Mumo, Amy; Majumdar, Himani; Pinter, Mary; Tuchman, Mendel; Krufka, Alison

    2014-01-01

    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 Kmapp for acetyl coenzyme A increased while the Kmapp 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 physiological

  5. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    PubMed

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  6. Calpain-10 Activity Underlies Angiotensin II-Induced Aldosterone Production in an Adrenal Glomerulosa Cell Model

    PubMed Central

    Seremwe, Mutsa; Schnellmann, Rick G.

    2015-01-01

    Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant production of aldosterone results in the development and progression of diseases including hypertension and congestive heart failure; therefore, a complete understanding of aldosterone production is important for developing more effective treatments. Angiotensin II (AngII) regulates steroidogenesis, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta-EF-hands, which are involved in various cellular responses. We hypothesized that calpain, in particular calpain-10, is activated by AngII in adrenal glomerulosa cells and underlies aldosterone production. Our studies showed that pan-calpain inhibitors reduced AngII-induced aldosterone production in 2 adrenal glomerulosa cell models, primary bovine zona glomerulosa and human adrenocortical carcinoma (HAC15) cells, as well as CYP11B2 expression in the HAC15 cells. Although AngII induced calpain activation in these cells, typical calpain inhibitors had no effect on AngII-elicited aldosterone production, suggesting a lack of involvement of classical calpains in this process. However, an inhibitor of the atypical calpain, calpain-10, decreased AngII-induced aldosterone production. Consistent with this result, small interfering RNA (siRNA)-mediated knockdown of calpain-10 inhibited aldosterone production and CYP11B2 expression, whereas adenovirus-mediated overexpression of calpain-10 resulted in increased AngII-induced aldosterone production. Our results indicate that AngII-induced activation of calpain-10 in glomerulosa cells underlies aldosterone production and identify calpain-10 or its downstream pathways as potential targets for the development of drug therapies for the treatment of hypertension. PMID:25836666

  7. Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).

    PubMed

    Pua, Eng-Chong; Chandramouli, Sumana; Han, Ping; Liu, Pei

    2003-01-01

    Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

  8. Functional regulation of neuronal nitric oxide synthase expression and activity in the rat retina.

    PubMed

    Walter, Lais Takata; Higa, Guilherme Shigueto Vilar; Schmeltzer, Christian; Sousa, Erica; Kinjo, Erika Reime; Rüdiger, Sten; Hamassaki, Dânia Emi; Cerchiaro, Giselle; Kihara, Alexandre Hiroaki

    2014-11-01

    In the nervous system within physiological conditions, nitric oxide (NO) production depends on the activity of nitric oxide synthases (NOSs), and particularly on the expression of the neuronal isoform (nNOS). In the sensory systems, the role of NO is poorly understood. In this study, we identified nNOS-positive cells in the inner nuclear layer (INL) of the rat retina, with distinct characteristics such as somata size, immunolabeling level and location. Employing mathematical cluster analysis, we determined that nNOS amacrine cells are formed by two distinct populations. We next investigated the molecular identity of these cells, which did not show colocalization with calbindin (CB), choline acetyltransferase (ChAT), parvalbumin (PV) or protein kinase C (PKC), and only partial colocalization with calretinin (CR), revealing the accumulation of nNOS in specific amacrine cell populations. To access the functional, circuitry-related roles of these cells, we performed experiments after adaptation to different ambient light conditions. After 24h of dark-adaptation, we detected a subtle, yet statistically significant decrease in nNOS transcript levels, which returned to steady-state levels after 24h of normal light-dark cycle, revealing that nNOS expression is governed by ambient light conditions. Employing electron paramagnetic resonance (EPR), we demonstrated that dark-adaptation decreases NO production in the retina. Furthermore, nNOS accumulation changed in the dark-adapted retinas, with a general reduction in the inner plexiform layer. Finally, computational analysis based on clustering techniques revealed that dark-adaptation differently affected both types of nNOS-positive amacrine cells. Taken together, our data disclosed functional regulation of nNOS expression and activity, disclosing new circuitry-related roles of nNOS-positive cells. More importantly, this study indicated unsuspected roles for NO in the sensory systems, particularly related to adaptation to

  9. Regulation of inducible nitric oxide synthase expression in beta cells by environmental factors: heavy metals.

    PubMed Central

    Eckhardt, W; Bellmann, K; Kolb, H

    1999-01-01

    The expression of inducible NO synthase (iNOS) in pancreatic islet beta cells modulates endocrine cell functions and, at very high levels of NO production causes beta-cell death. We tested the hypothesis that environmental factors such as heavy-metal salts modulate iNOS expression in beta cells. A rat beta-cell line (insulinoma RINm5F) was cultured in the presence of low-dose interleukin (IL)-1beta for suboptimal induction of iNOS. PbCl2 (0. 1-10 microM) dose-dependently increased NO (measured as nitrite) formation (P<0.001). In contrast, HgCl2 suppressed nitrite production (0.1-10 microM, P<0.05). Measurements of iNOS activity by determining citrulline levels confirmed the potentiating effect of PbCl2 (P<0.05). There was a narrow time window of heavy-metal actions, ranging from -24 h (Hg2+) or -3 h (Pb2+) to +2 h, relative to the addition of IL-1beta. By semi-quantitative reverse transcriptase-PCR, enhanced levels of iNOS mRNA were found in the presence of Pb2+ (P<0.05) and decreased levels in the presence of Hg2+. The amount of iNOS protein as determined by Western blotting was increased in the presence of Pb2+. We conclude that Pb2+ upregulates and Hg2+ suppresses iNOS gene expression at the level of transcription, probably by acting on the signalling pathway. These observations may have important implications for understanding pathological effects of environmental factors on endocrine organ functions. PMID:10051441

  10. Interleukin-13 inhibits inducible nitric oxide synthase expression in human mesangial cells.

    PubMed Central

    Saura, M; Martínez-Dalmau, R; Minty, A; Pérez-Sala, D; Lamas, S

    1996-01-01

    The synthesis of nitric oxide in inflammatory situations requires the expression of an inducible isoform of nitric oxide synthase (iNOS). Human mesangial cells (HMC) express an iNOS enzyme after exposure to multiple co-stimuli. In this study we have observed that while tumour necrosis factor-alpha, interleukin (IL)-1 beta, interferon-gamma and bacterial lipopolysaccharide (LPS) were unable to significantly induce NO synthesis when used alone, they induced an evident stimulation of NO synthesis when used in various combinations. A mixture of the three cytokines (CM) and LPS resulted in a 10-15-fold stimulation of NO synthesis over control values which started to be significant after 16 h. The addition of IL-13, a cytokine with anti-inflammatory properties, inhibited CM/LPS-induced NO synthesis in a concentration-dependent manner. A marked inhibitory effect (60-65%) could be observed when HMC were treated with IL-13 (10 ng/ml) 24 h before, at the same time as, or even 4 h after the addition of CM/LPS. This inhibitory effect was still significant (25%) when IL-13 was added 16 h after CM/LPS. Northern analysis showed that IL-13-mediated iNOS inhibition was closely correlated with the suppression of iNOS mRNA expression. These results identify IL-13 as a powerful regulatory tool for the inhibition of NO synthesis in human cells, a property which may be pathophysiologically relevant in NO-related inflammatory processes. PMID:8573104

  11. Fatty Acid Synthase Polymorphisms, Tumor Expression, Body Mass Index, Prostate Cancer Risk, and Survival

    PubMed Central

    Nguyen, Paul L.; Ma, Jing; Chavarro, Jorge E.; Freedman, Matthew L.; Lis, Rosina; Fedele, Giuseppe; Fiore, Christopher; Qiu, Weiliang; Fiorentino, Michelangelo; Finn, Stephen; Penney, Kathryn L.; Eisenstein, Anna; Schumacher, Fredrick R.; Mucci, Lorelei A.; Stampfer, Meir J.; Giovannucci, Edward; Loda, Massimo

    2010-01-01

    Purpose Fatty acid synthase (FASN) regulates de novo lipogenesis, body weight, and tumor growth. We examined whether common germline single nucleotide polymorphisms (SNPs) in the FASN gene affect prostate cancer (PCa) risk or PCa-specific mortality and whether these effects vary by body mass index (BMI). Methods In a prospective nested case-control study of 1,331 white patients with PCa and 1,267 age-matched controls, we examined associations of five common SNPs within FASN (and 5 kb upstream/downstream, R2 > 0.8) with PCa incidence and, among patients, PCa-specific death and tested for an interaction with BMI. Survival analyses were repeated for tumor FASN expression (n = 909). Results Four of the five SNPs were associated with lethal PCa. SNP rs1127678 was significantly related to higher BMI and interacted with BMI for both PCa risk (Pinteraction = .004) and PCa mortality (Pinteraction = .056). Among overweight men (BMI ≥ 25 kg/m2), but not leaner men, the homozygous variant allele carried a relative risk of advanced PCa of 2.49 (95% CI, 1.00 to 6.23) compared with lean men with the wild type. Overweight patients carrying the variant allele had a 2.04 (95% CI, 1.31 to 3.17) times higher risk of PCa mortality. Similarly, overweight patients with elevated tumor FASN expression had a 2.73 (95% CI, 1.05 to 7.08) times higher risk of lethal PCa (Pinteraction = .02). Conclusion FASN germline polymorphisms were significantly associated with risk of lethal PCa. Significant interactions of BMI with FASN polymorphisms and FASN tumor expression suggest FASN as a potential link between obesity and poor PCa outcome and raise the possibility that FASN inhibition could reduce PCa-specific mortality, particularly in overweight men. PMID:20679621

  12. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  13. An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.

    PubMed

    Ampasala, Dinakar R; Zheng, Sichun; Zhang, Dayu; Ladd, Tim; Doucet, Daniel; Krell, Peter J; Retnakaran, Arthur; Feng, Qili

    2011-02-01

    Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect.

  14. Expression of thromboxane synthase and the thromboxane-prostanoid receptor in the mouse and rat retina

    PubMed Central

    Wright, William S.; McElhatten, Robert M.; Harris, Norman R.

    2009-01-01

    Experimental models of the diabetic retina have suggested a pathological role for thromboxane. To date however, little information is available as to the cellular locations of retinal thromboxane synthase (TxS), or its receptor, even in non-diabetic controls. In this study, C57BL/6 mice and Wistar rats were injected with streptozotocin to induce diabetes, or with buffer for non-diabetic controls. Four weeks following the injection, eyes were enucleated and labeled for TxS and the thromboxane-prostanoid (TP) receptor. Immunofluorescent intensity was quantified in the ganglion cell plus inner plexiform layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor inner segment. Even in control mice and rats, all layers of the retina showed immunoreactivity for TxS and the TP receptor: however, the pattern of expression demonstrated an inverse relationship, with the highest TxS staining in the inner retina, and the highest TP receptor staining in the outer retina (more specifically, in the photoreceptor inner segment). Four weeks of hyperglycemia did not increase the retinal levels of TxS or TP receptor; however, TP receptor intensities in the outer retina of diabetic rats were highly variable (mostly high but some low), with no values from the photoreceptor inner segment in the same range as obtained from controls. PMID:19523949

  15. Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit.

    PubMed

    Aluru, Maneesha R; Mazourek, Michael; Landry, Laurie G; Curry, Jeanne; Jahn, Molly; O'Connell, Mary A

    2003-07-01

    The biosynthesis of capsaicinoids in the placenta of chilli fruit is modelled to require components of the fatty acid synthase (FAS) complex. Three candidate genes for subunits in this complex, Kas, Acl, and Fat, isolated based on differential expression, were characterized. Transcription of these three genes was placental-specific and RNA abundance was positively correlated with degree of pungency. Kas and Acl were mapped to linkage group 1 and Fat to linkage group 6. None of the genes is linked to the pungency locus, C, on linkage group 2. KAS accumulation was positively correlated with pungency. Western blots of placental extracts and histological sections both demonstrated that the accumulation of this enzyme was correlated with fruit pungency and KAS was immunolocalized to the expected cell layer, the placental epidermis. Enzyme activity of the recombinant form of the placental-specific KAS was confirmed using crude cell extracts. These FAS components are fruit-specific members of their respective gene families. These genes are predicted to be associated with Capsicum fruit traits, for example, capsaicinoid biosynthesis or fatty acid biosynthesis necessary for placental development.

  16. Expression of nitric oxide synthase in the developing eye of Zebrafish Danio rerio

    NASA Astrophysics Data System (ADS)

    Wang, Yongjun; Zhang, Shicui; Sawant, M. S.

    2004-12-01

    Expression of nitric oxide synthase (NOS) in the developing eye of zebrafish was studied by NADPH-diaphorase staining technique. NOS activity was first observed in the optic primordium and the lens placode at 5-somite stage, and remained basically unchanged up to the prim-5 stage. Upon hatching, NOS activity was nearly equally detected in the gangalion cell layer and the photoreceptor layer in the developing retina. However, it began declining in the inner plexiform layer and the inner nuclear layer at this stage. NOS activity disappeared in the lens although the anterior lens epithelium was strongly stained. Two days after hatching, NOS activity was still strong in the photoreceptor layer, but decreased markedly in the gangalion cell layer, the inner plexiform layer and the inner nuclear layer with the retinal patterning. These suggested that nitric oxide (NO), the product of NOS, is not only involved in the modulation of patterning and differentiation of the retinal cells but also in the regulation of proliferation, and differentiation of the lens fibrocytes.

  17. Interleukin-18 deficiency protects against renal interstitial fibrosis in aldosterone/salt-treated mice.

    PubMed

    Tanino, Akiko; Okura, Takafumi; Nagao, Tomoaki; Kukida, Masayoshi; Pei, Zuowei; Enomoto, Daijiro; Miyoshi, Ken-Ichi; Okamura, Haruki; Higaki, Jitsuo

    2016-10-01

    Interleukin (IL)-18 is a member of the IL-1 family of cytokines and was described originally as an interferon γ-inducing factor. Aldosterone plays a central role in the regulation of sodium and potassium homoeostasis by binding to the mineralocorticoid receptor and contributes to kidney and cardiovascular damage. Aldosterone has been reported to induce IL-18, resulting in cardiac fibrosis with induced IL-18-mediated osteopontin (OPN). We therefore hypothesized that aldosterone-induced renal fibrosis via OPN may be mediated by IL-18. To verify this hypothesis, we compared mice deficient in IL-18 and wild-type (WT) mice in a model of aldosterone/salt-induced hypertension. IL-18(-/-) and C57BL/6 WT mice were used for the uninephrectomized aldosterone/salt hypertensive model, whereas NRK-52E cells (rat kidney epithelial cells) were used in an in vitro model. In the present in vivo study, IL-18 protein expression was localized in medullary tubules in the WT mice, whereas in aldosterone-infused WT mice this expression was up-regulated markedly in the proximal tubules, especially in injured and dilated tubules. This renal damage caused by aldosterone was attenuated significantly by IL-18 knockout with down-regulation of OPN expression. In the present in vitro study, aldosterone directly induced IL-18 gene expression in renal tubular epithelial cells in a concentration- and time-dependent manner. These effects were inhibited completely by spironolactone. IL-18 may be a key mediator of aldosterone-induced renal fibrosis by inducing OPN, thereby exacerbating renal interstitial fibrosis. Inhibition of IL-18 may therefore provide a potential target for therapeutic intervention aimed at preventing the progression of renal injury.

  18. Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during Giardia lamblia infection.

    PubMed

    Maloney, Jenny; Keselman, Aleksander; Li, Erqiu; Singer, Steven M

    2015-06-01

    Nitric oxide (NO) has been shown to inhibit Giardia lamblia in vitro and in vivo. This study sought to determine if Giardia infection induces arginase 1 (ARG1) expression in host macrophages to reduce NO production. Stimulations of RAW 264.7 macrophage-like cells with Giardia extract induced arginase activity. Real-time PCR and immunohistochemistry showed increased ARG1 and nitric oxide synthase 2 (NOS2) expression in mouse intestine following infection. Flow cytometry demonstrated increased numbers of macrophages positive for both ARG1 and NOS2 in lamina propria following infection, but there was no evidence of increased expression of ARG1 in these cells.

  19. Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells.

    PubMed Central

    Recklies, A D; White, C; Melching, L; Roughley, P J

    2001-01-01

    Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-beta1) and cytokines [interleukin 1beta1 (IL-1beta)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1beta, and showed a unique synergistic response when IL-1beta was used in combination with TGF-beta1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-beta1/IL-1beta, the magnitude of the change was far less than the effect on HA synthesis. Our data thus

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

    SciTech Connect

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. )

    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.

  1. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

    PubMed Central

    Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

    2016-01-01

    Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

  2. Sleep and Serotonin Modulate Paracapsular Nitric Oxide Synthase Expressing Neurons of the Amygdala

    PubMed Central

    Fisher, Simon P.; Ellender, Tommas J.; Vyazovskiy, Vladyslav V.

    2016-01-01

    Abstract Unraveling the roles of distinct neuron types is a fundamental challenge to understanding brain function in health and disease. In the amygdala, a brain structure regulating emotional behavior, the diversity of GABAergic neurons has been only partially explored. We report a novel population of GABAergic amygdala neurons expressing high levels of neuronal nitric oxide synthase (nNOS). These cells are predominantly localized along basolateral amygdala (BLA) boundaries. Performing ex vivo patch-clamp recordings from nNOS+ neurons in Nos1-CreER;Ai9 mice, we observed that nNOS+ neurons located along the external capsule display distinctive electrophysiological properties, axonal and dendritic arborization, and connectivity. Examining their c-Fos expression, we found that paracapsular nNOS+ neurons are activated during a period of undisturbed sleep following sleep deprivation, but not during sleep deprivation. Consistently, we found that dorsal raphe serotonin [5-hydroxytryptamine (5-HT)] neurons, which are involved in sleep–wake regulation, innervate nNOS+ neurons. Bath application of 5-HT hyperpolarizes nNOS+ neurons via 5-HT1A receptors. This hyperpolarization produces a reduction in firing rate and, occasionally, a switch from tonic to burst firing mode, thereby contrasting with the classic depolarizing effect of 5-HT on BLA GABAergic cells reported so far. Thus, nNOS+ cells are a distinct cell type of the amygdala that controls the activity of downstream neurons in both amygdaloid and extra-amygdaloid regions in a vigilance state-dependent fashion. Given the strong links among mood, sleep deprivation, and 5-HT, the recruitment of paracapsular nNOS+ neurons following high sleep pressure may represent an important mechanism in emotional regulation. PMID:27822504

  3. Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation.

    PubMed

    Baskurt, Oguz K; Yalcin, Ozlem; Ozdem, Sadi; Armstrong, Jonathan K; Meiselman, Herbert J

    2004-01-01

    The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.

  4. Phylogenetic and expression analysis of sucrose phosphate synthase isozymes in plants.

    PubMed

    Lutfiyya, Linda L; Xu, Nanfei; D'Ordine, Robert L; Morrell, James A; Miller, Philip W; Duff, Stephen M G

    2007-07-01

    In plants and microbes, sucrose phosphate synthase (SPS) is an important enzyme in sucrose biosynthesis. Several different isozymes of SPS exist in plants. Genomic and EST sequence data from Arabidopsis, rice and maize has been analyzed. This analysis has revealed that the Arabidopsis genome contains four unique SPS genes. The rice databases (Monsanto proprietary, and public databases) contain five unique full-length SPS genes. Using the Monsanto maize EST and genomic sequence databases, we have identified five full length and two partial SPS sequences, bringing the total number of presently known maize SPS genes to at least seven. Phylogenetic analysis of all known SPS sequences revealed several putative evolutionary branches of SPS. We have classified SPS genes into three major groups in higher plants, all with distinct features from the known microbial SPS genes. Furthermore, this analysis suggests evolutionary divergence of monocotyledonous (monocot) and dicotyledonous (dicot) SPS sequences. The evidence suggests that several gene duplication events occurred at various points during evolution, both before and after the monocot/dicot split. It appears that at least one of the major forms of SPS genes may have evolved after the divergence of monocots and dicots. In addition, several more recent gene duplication events may have occurred after maize/rice speciation, giving rise to additional SPS genes in maize. Some of the variants lack one or more of the presently known regulatory sites, implying that this evolutionary divergence may have given rise to enzymes with functional differences. We present evidence from transcript distribution studies using cDNA libraries as well as transcriptional profiling experiments and propose that specific SPS genes have diverse patterns of expression that are sometimes responsive to environmental signals. Our data suggests that higher plant SPS isozymes differ with respect to their patterns of expression and regulation and that

  5. Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats

    PubMed Central

    Martín-Fernández, Beatriz; Rubio-Navarro, Alfonso; Cortegano, Isabel; Ballesteros, Sandra; Alía, Mario; Cannata-Ortiz, Pablo; Olivares-Álvaro, Elena; Egido, Jesús; de Andrés, Belén; Gaspar, María Luisa; de las Heras, Natalia; Lahera, Vicente; Moreno, Juan Antonio

    2016-01-01

    We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt–treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt–treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation. PMID:26730742

  6. Interleukin-33/ST2 system attenuates aldosterone-induced adipogenesis and inflammation.

    PubMed

    Martínez-Martínez, Ernesto; Cachofeiro, Victoria; Rousseau, Elodie; Álvarez, Virginia; Calvier, Laurent; Fernández-Celis, Amaya; Leroy, Céline; Miana, María; Jurado-López, Raquel; Briones, Ana M; Jaisser, Frederic; Zannad, Faiez; Rossignol, Patrick; López-Andrés, Natalia

    2015-08-15

    Interleukin-33 (IL-33) but not soluble ST2 (sST2) exerts anti-inflammatory and protective effects in several tissues. Aldosterone, a proinflammatory mediator which promotes adipogenesis, is elevated in obese patients. The aim of this study was to investigate the interactions between IL-33/ST2 system and Aldosterone in adipose tissue. Rats fed a high fat diet presented increased sST2 expression, diminished IL-33/sST2 ratio and enhanced levels of differentiation and inflammation in adipose tissue as compared to controls. A similar pattern was observed in adipose tissue from C57BL/6 Aldosterone-treated mice. In both animal models, Aldosterone was correlated with sST2. Treatment of 3T3-L1 adipocytes with IL-33 delayed adipocyte differentiation diminished lipid accumulation and decreased inflammation. Aldosterone decreased IL-33 and increased sST2 expressions in differentiated adipocytes. Aldosterone-induced adipocyte differentiation and inflammation were blocked by IL-33 treatment, but sST2 did not exert any effects. The crosstalk between IL-33/ST2 and Aldosterone could be relevant in the metabolic consequences of obesity.

  7. A Cytokine Signalling Network for the Regulation of Inducible Nitric Oxide Synthase Expression in Rheumatoid Arthritis

    PubMed Central

    Dey, Poulami; Panga, Venugopal; Raghunathan, Srivatsan

    2016-01-01

    In rheumatoid arthritis (RA), nitric oxide (NO) is implicated in inflammation, angiogenesis and tissue destruction. The enzyme inducible nitric oxide synthase (iNOS) is responsible for the localised over-production of NO in the synovial joints affected by RA. The pro- and anti-inflammatory cytokines stimulate the synovial macrophages and the fibroblast-like synoviocytes to express iNOS. Therefore, the cytokine signalling network underlying the regulation of iNOS is essential to understand the pathophysiology of the disease. By using information from the literature, we have constructed, for the first time, the cytokine signalling network involved in the regulation of iNOS expression. Using the differential expression patterns obtained by re-analysing the microarray data on the RA synovium and the synovial macrophages available in the Gene Expression Omnibus (GEO) database, we aimed to establish the role played by the network genes towards iNOS regulation in the RA synovium. Our analysis reveals that the network genes belonging to interferon (IFN) and interleukin-10 (IL-10) pathways are always up-regulated in the RA synovium whereas the genes which are part of the anti-inflammatory transforming growth factor-beta (TGF-β) signalling pathway are mostly down-regulated. We observed a consistent up-regulation of the transcription factor signal transducers and activators of transcription 1 (STAT1) in the RA synovium and the macrophages. Interestingly, we found a consistent up-regulation of the iNOS interacting protein ras-related C3 botulinum toxin substrate 2 (RAC2) in the RA synovium as well as the macrophages. Importantly, we have constructed a model to explain the impact of IFN and IL-10 pathways on Rac2-iNOS interaction leading to over-production of NO and thereby causing chronic inflammation in the RA synovium. The interplay between STAT1 and RAC2 in the regulation of NO could have implications for the identification of therapeutic targets for RA. PMID:27626941

  8. High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

    PubMed Central

    Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

    2014-01-01

    Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

  9. Cyclosporin-A inhibits constitutive nitric oxide synthase activity and neuronal and endothelial nitric oxide synthase expressions after spinal cord injury in rats.

    PubMed

    Diaz-Ruiz, Araceli; Vergara, Paula; Perez-Severiano, Francisca; Segovia, Jose; Guizar-Sahagún, Gabriel; Ibarra, Antonio; Ríos, Camilo

    2005-02-01

    Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca2+/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.

  10. Homologous and heterologous expression of grapevine E-(β)-caryophyllene synthase (VvGwECar2).

    PubMed

    Salvagnin, Umberto; Carlin, Silvia; Angeli, Sergio; Vrhovsek, Urska; Anfora, Gianfranco; Malnoy, Mickael; Martens, Stefan

    2016-11-01

    E-(β)-caryophyllene is a sesquiterpene volatile emitted by plants and involved in many ecological interactions within and among trophic levels and it has a kairomonal activity for many insect species. In grapevine it is a key compound for host-plant recognition by the European grapevine moth, Lobesia botrana, together with other two sesquiterpenes. In grapevine E-(β)-caryophyllene synthase is coded by the VvGwECar2 gene, although complete characterization of the corresponding protein has not yet been achieved. Here we performed the characterization of the enzyme after heterologous expression in E. coli, which resulted to produce in vitro also minor amounts of the isomer α-humulene and of germacrene D. The pH optimum was estimated to be 7.8, and the Km and Kcat values for farnesyl pyrophosphate were 31.4 μM and 0.19 s(-1) respectively. Then, we overexpressed the gene in the cytoplasm of two plant species, Arabidopsis thaliana and the native host Vitis vinifera. In Arabidopsis the enzyme changed the plant head space release, showing a higher selectivity for E-(β)-caryophyllene, but also the production of thujopsene instead of germacrene D. Overall plants increased the E-(β)-caryophyllene emission in the headspace collection by 8-fold compared to Col-0 control plants. In grapevine VvGwECar2 overexpression resulted in higher E-(β)-caryophyllene emissions, although there was no clear correlation between gene activity and sesquiterpene quantity, suggesting a key role by the plant regulation machinery.

  11. Cloning, expression and characterization of an insect geranylgeranyl diphosphate synthase from Choristoneura fumiferana.

    PubMed

    Barbar, Aline; Couture, Manon; Sen, Stephanie E; Béliveau, Catherine; Nisole, Audrey; Bipfubusa, Marie; Cusson, Michel

    2013-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.

  12. Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005.

    PubMed

    Gao, Yun; Xi, Yue; Lu, Xiao-Ling; Zheng, Heng; Hu, Bo; Liu, Xiao-Yu; Jiao, Bing-Hua

    2013-11-01

    Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k cat/K m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [(2)H2]trehalose combined with unlabeled trehalose was converted to [(2)H2]maltose and maltose, but without any production of [(2)H]maltose or [(2)H]trehalose and with no incorporation of exogenous [(2)H7]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.

  13. Aldosterone-induced brain MAPK signaling and sympathetic excitation are angiotensin II type-1 receptor dependent.

    PubMed

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Felder, Robert B

    2012-02-01

    Angiotensin II (ANG II)-induced mitogen-activated protein kinase (MAPK) signaling upregulates angiotensin II type-1 receptors (AT(1)R) in hypothalamic paraventricular nucleus (PVN) and contributes to AT(1)R-mediated sympathetic excitation in heart failure. Aldosterone has similar effects to increase AT(1)R expression in the PVN and sympathetic drive. The present study was undertaken to determine whether aldosterone also activates the sympathetic nervous system via MAPK signaling and, if so, whether its effect is independent of ANG II and AT(1)R. In anesthetized rats, a 4-h intravenous infusion of aldosterone induced increases (P < 0.05) in phosphorylated (p-) p44/42 MAPK in PVN, PVN neuronal excitation, renal sympathetic nerve activity (RSNA), mean blood pressure (MBP), and heart rate (HR). Intracerebroventricular or bilateral PVN microinjection of the p44/42 MAPK inhibitor PD-98059 reduced the aldosterone-induced RSNA, HR, and MBP responses. Intracerebroventricular pretreatment (5 days earlier) with pooled small interfering RNAs targeting p44/42 MAPK reduced total and p-p44/42 MAPK, aldosterone-induced c-Fos expression in the PVN, and the aldosterone-induced increases in RSNA, HR, and MBP. Intracerebroventricular infusion of either the mineralocorticoid receptor antagonist RU-28318 or the AT(1)R antagonist losartan blocked aldosterone-induced phosphorylation of p44/42 MAPK and prevented the increases in RSNA, HR, and MBP. These data suggest that aldosterone-induced sympathetic excitation depends upon that AT(1)R-induced MAPK signaling in the brain. The short time course of this interaction suggests a nongenomic mechanism, perhaps via an aldosterone-induced transactivation of the AT(1)R as described in peripheral tissues.

  14. cDNA cloning, expression, and characterization of Taro SSII: a novel member of starch synthase II family.

    PubMed

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-05

    A novel soluble starch synthase II (SSII) gene was isolated from taro (Colocasia esculenta var. esculenta) tubers. This 2939 bp SSII transcript encodes 804 amino acids with a putative transit peptide of 52 residues. It displays 58-63% identity and 63-69% similarity with SSIIs from other sources. Alignment and phylogenetic analyses showed that taro SSII is more closely related with dicot SSIIs than with the monocot ones, though taro is a monocot. The identification of taro SSII clone as starch synthase was confirmed by the expression of its enzymatic activity in Escherichia coli. Genomic DNA blot analysis revealed a single copy or low number copies of SSII in taro. Expression profile showed that more transcript and protein were accumulated in tubers of 597 +/- 37 g fresh weight, that is, a stage of rapid starch synthesis, than tubers of other stages. By Western blot analysis, SSII was found in both soluble and granule bound portions of tuber extracts, and more SSII protein was found in aged leaves than in leaves of other stages. These results suggest that taro SSII is a novel starch synthase for the synthesis of both transit and storage starch.

  15. Ménage à trois: aldosterone, sodium and nitric oxide in vascular endothelium.

    PubMed

    Fels, Johannes; Oberleithner, Hans; Kusche-Vihrog, Kristina

    2010-12-01

    Aldosterone, a mineralocorticoid hormone mainly synthesized in the adrenal cortex, has been recognized to be a regulator of cell mechanics. Recent data from a number of laboratories implicate that, besides kidney, the cardiovascular system is an important target for aldosterone. In the endothelium, it promotes the expression of epithelial sodium channels (ENaC) and modifies the morphology of cells in terms of mechanical stiffness, surface area and volume. Additionally, it renders the cells highly sensitive to small changes in extracellular sodium and potassium. In this context, the time course of aldosterone action is pivotal. In the fast (seconds to minutes), non-genomic signalling pathway vascular endothelial cells respond to aldosterone with transient swelling, softening and insertion of ENaC in the apical plasma membrane. In parallel, nitric oxide (NO) is released from the cells. In the long-term (hours), aldosterone has opposite effects: The mechanical stiffness increases, the cells shrink and NO production decreases. This leads to the conclusion that both the physiology and pathophysiology of aldosterone action in the vascular endothelium are closely related. Aldosterone, at concentrations in the physiological range and over limited time periods can stabilize blood pressure and regulate tissue perfusion while chronically high concentrations of this hormone over extended time periods impair sodium homeostasis promoting endothelial dysfunction and the development of tissue fibrosis.

  16. Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes.

    PubMed Central

    Neidle, E L; Kaplan, S

    1993-01-01

    The nucleotide sequences of the Rhodobacter sphaeroides hemA and hemT genes, encoding 5-aminolevulinic acid (ALA) synthase isozymes, were determined. ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A, the first and rate-limiting step in tetrapyrrole biosynthesis. The hemA and hemT structural gene sequences were 65% identical to each other, and the deduced HemA and HemT polypeptide sequences were 53% identical, with an additional 16% of aligned amino acids being similar. HemA and HemT were homologous to all characterized ALA synthases, including two human ALA synthase isozymes. In addition, they were evolutionarily related to 7-keto-8-aminopelargonic acid synthetase (BioF) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), enzymes which catalyze similar reactions. Two hemA transcripts were identified, both expressed under photosynthetic conditions at levels approximately three times higher than those found under aerobic conditions. A single transcriptional start point was identified for both transcripts, and a consensus sequence at this location indicated that an Fnr-like protein may be involved in the transcriptional regulation of hemA. Transcription of hemT was not detected in wild-type cells under the physiological growth conditions tested. In a mutant strain in which the hemA gene had been inactivated, however, hemT was expressed. In this mutant, hemT transcripts were characterized by Northern (RNA) hybridization, primer extension, and ribonuclease protection techniques. A small open reading frame of unknown function was identified upstream of, and transcribed in the same direction as, hemA. Images PMID:8468290

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

    SciTech Connect

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh . E-mail: rakbhat01@yahoo.com

    2005-10-14

    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 {sup {omega}}-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

  18. Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets

    PubMed Central

    Luo, P.; Kreger, M. T.; Brissova, M.; Dai, C.; Whitfield, T. T.; Kim, H. S.; Wasserman, D. H.; Powers, A. C.; Brown, N. J.

    2011-01-01

    Aims/hypothesis Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. Methods We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2]−/−)and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As−/− mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. Results Fasting glucose concentrations were reduced in As−/−mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As−/− than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As−/− mice even during high sodium intake. There was no difference in insulin sensitivity between As−/− and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. Conclusions/interpretation We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess. PMID:21519965

  19. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  20. Sequential changes in redox status and nitric oxide synthases expression in the liver after bile duct ligation.

    PubMed

    Vázquez-Gil, M José; Mesonero, M José; Flores, Olga; Criado, Manuela; Hidalgo, Froilán; Arévalo, Miguel A; Sánchez-Rodríguez, Angel; Tuñón, M Jesús; López-Novoa, José M; Esteller, A

    2004-06-25

    Bile duct ligation (BDL) in rats induces portal fibrosis. This process has been linked to changes in the oxidative state of the hepatic cells and in the production of nitric oxide. Our objective was to find possible temporal connections between hepatic redox state, NO synthesis and liver injury. In this work we have characterized hepatic lesions 17 and 31 days after BDL and determined changes in hepatic function, oxidative state, and NO production. We have also analyzed the expression and localization of inducible NO synthase (NOS2) and constitutive NO synthase (NOS3). After 17 and 31 days from ligature, lipid peroxidation is increased and both plasma concentration and biliary excretion of nitrite+nitrate are rised. 17 days after BDL both NOS2 and NOS3 are expressed intensely and in the same regions. 31 days after BDL, the expression of NOS2 remains elevated and is localized mostly in preserved hepatocytes in portal areas and in neighborhoods of centrolobulillar vein. NOS3 is localized in vascular regions of portal spaces and centrolobulillar veins and in preserved sinusoids and although its expression is greater than in control animals (34%), it is clearly lower (50%) than 17 days after BDL. The time after BDL is crucial in the study of NO production, intrahepatic localization of NOS isoforms expression, and cell type involved, since all these parameters change with time. BDL-induced, peroxidation and fibrosis are not ligated by a cause-effect relationship, but rather they both seem to be the consequence of common inductors.

  1. Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

    SciTech Connect

    Huynh, Frederick; Tan, Tien-Chye; Swaminathan, Kunchithapadam; Patel, Bharat K. C.

    2005-01-01

    The first crystallographic study of a sucrose phosphate synthase from H. orenii, an organism that is both thermophilic and halophilic, is reported. The protein crystal diffracts X-rays to 3.01 Å. This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure.

  2. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera

    PubMed Central

    Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S.

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides. PMID:26919744

  3. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

    PubMed Central

    Hirsch, J P; Henry, S A

    1986-01-01

    The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression. Images PMID:3025587

  4. Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

    PubMed Central

    Oteiza, Patricia I.; Link, Samuel; Hey, Valentin; Stopper, Helga; Schupp, Nicole

    2014-01-01

    Abstract Aims: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. Results: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. Innovation: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. Conclusion: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects. Antioxid. Redox Signal. 21, 2126–2142. PMID:24512358

  5. HUMAN INTERVENTIONS TO CHARACTERIZE NOVEL RELATIONSHIPS BETWEEN THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM AND PARATHYROID HORMONE

    PubMed Central

    Brown, Jenifer M.; Williams, Jonathan S.; Luther, James M.; Garg, Rajesh; Garza, Amanda E.; Pojoga, Luminita H.; Ruan, Daniel T.; Williams, Gordon H.; Adler, Gail K.; Vaidya, Anand

    2014-01-01

    Observational studies in primary hyperaldosteronism (PA) suggest a positive relationship between aldosterone and parathyroid hormone (PTH); however, interventions to better characterize the physiologic relationship between the renin-angiotensin-aldosterone system (RAAS) and PTH are needed. We evaluated the effect of individual RAAS components on PTH using 4 interventions in humans without PA. PTH was measured before and after: Study 1) low-dose angiotensin II [AngII] infusion (1 ng/kg/min) and captopril administration (25 mg × 1); Study 2) high-dose AngII infusion (3 ng/kg/min); Study 3) blinded crossover randomization to aldosterone infusion (0.7 µg/kg/hr) and vehicle; and Study 4) blinded randomization to spironolactone (50mg/daily) or placebo for 6 weeks. Infusion of AngII at 1 ng/kg/min acutely increased aldosterone (+148%) and PTH (+10.3%), while AngII at 3 ng/kg/min induced larger incremental changes in aldosterone (+241%) and PTH (+36%) (P<0.01). Captopril acutely decreased aldosterone (−12%) and PTH (−9.7%) (P<0.01). In contrast, aldosterone infusion robustly raised serum aldosterone (+892%) without modifying PTH. However, spironolactone therapy over 6 weeks modestly lowered PTH when compared to placebo (P<0.05). In vitro studies revealed the presence of AngII type I and mineralocorticoid receptor mRNA and protein expression in normal and adenomatous human parathyroid tissues. We observed novel pleiotropic relationships between RAAS components and the regulation of PTH in individuals without PA: the acute modulation of PTH by the RAAS appears to be mediated by AngII, whereas the long-term influence of the RAAS on PTH may involve aldosterone. Future studies to evaluate the impact of RAAS inhibitors in treating PTH-mediated disorders are warranted. PMID:24191286

  6. Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

    PubMed Central

    Bohlmann, Jörg; Crock, John; Jetter, Reinhard; Croteau, Rodney

    1998-01-01

    (E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production. PMID:9618485

  7. Elevation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in the Mouse Brain after Chronic Nonylphenol Exposure

    PubMed Central

    Zhang, Yan-Qiu; Mao, Zhen; Zheng, Yuan-Lin; Han, Bao-Ping; Chen, Ling-Tong; Li, Jing; Li, Fei

    2008-01-01

    The present study was performed to investigate the effects of chronic administration of nonylphenol (NP) on the expression of inflammation-related genes in the brains of mice. NP was given orally by gavages at 0, 50, 100, and 200 mg/kg/d. The expression of inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was evaluated by immunohistochemistry and immunoblotting assays. The nitric oxide (NO) level and nitric oxide synthase (NOS) activity were also measured by biochemical analyses. The results showed that NP at a high dose (200 mg/kg/d) significantly increased the expression of iNOS and COX-2 in both the hippocampus and cortex. In parallel with the increase in iNOS expression, the NO level was significantly greater at the dose of 200 mg/kg/d, compared to the control. The activity of NOS was also increased in the brain of mice at the dose of 100 and 200 mg/kg/d. These findings demonstrate that NP may have the potential to induce the chronic inflammation or cause neurotoxicity in the mouse brain. PMID:19325730

  8. Ketogenic mitochondrial 3-hydroxy 3-methylglutaryl-CoA synthase gene expression in intestine and liver of suckling rats.

    PubMed

    Serra, D; Asins, G; Hegardt, F G

    1993-03-01

    The ketogenic mitochondrial 3-hydroxy 3-methylglutaryl-coenzyme A (HMG-CoA) synthase gene is expressed in intestine of suckling rats, its mRNA levels changing with age. Intestine mitochondrial mRNA values reach maximum levels on the 12th postnatal day and then decrease smoothly. Mother's milk may influence the intestine expression, since mRNA levels at birth are very low, increasing after the first lactation. Moreover, rats weaned at either Day 18 or 21 decrease their mRNA levels dramatically and there is no expression in adult rats. Mitochondrial HMG-CoA synthase is also expressed in liver of suckling rats but the developmental pattern of mRNAs is different from that in intestine, showing the highest values at Day 3 of life. mRNA levels in liver are lower than in intestine for most of the suckling period, suggesting the physiological relevance of the intestine for the ketogenic process of the whole body. Liver mRNA levels on weaning and in adult rats are high enough to sustain hepatic ketogenesis.

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

    PubMed

    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.

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

    PubMed Central

    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 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

  11. Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.).

    PubMed

    Yuan, Kejun; Wang, Changjun; Xin, Li; Zhang, Anning; Ai, Chengxiang

    2013-07-25

    A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar "White Winter Pearmain". When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4°C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.

  12. Analysis of THCA synthase gene expression in cannabis: a preliminary study by real-time quantitative PCR.

    PubMed

    Cascini, Fidelia; Passerotti, Stella; Boschi, Ilaria

    2013-09-10

    In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression. Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.

  13. [Primary aldosteronism and pregnancy: report of 2 cases].

    PubMed

    Germain, Alfredo M; Kottman, Cristián; Valdés, Gloria

    2002-12-01

    Based on two patients, we discuss the difficulties in diagnosing and managing primary aldosteronism in pregnancy, which derive from changes of the renin-angiotensin-aldosterone axis, from the uncertainty regarding blood pressure control along gestation and postpartum, and from the contraindication to the use of spironolactone. The first case is a 27 years old woman with a long standing refractory hypertension, a hemorrhagic stroke with left brachial hemiplegia and crural hemiparesia, two miscarriages, one stillbirth and one offspring with intrauterine growth retardation. Due to hypokalemia, a plasma aldosterone/renin activity ratio of 91, and a negative genetic screening for glucocorticoid remediable aldosteronism (GRA), a primary hyperaldosteronism with normal adrenals in CT scan was diagnosed, and good blood pressure control was attained with spironolactone. After two and a half years of normotension, a fifth pregnancy, managed with methyldopa evolved with satisfactory blood pressures, plasma potassium, fetal growth, uterine and umbilical arterial resistance indexes, and maternal endothelial function. At 37 1/2 weeks of pregnancy the patient delivered a healthy newborn weighing 2,960 g. Blood pressure rose during the 48 hours of postpartum in the absence of proteinuria and required i.v. hydralazine. The second patient is a 37 years old woman, with known refractory hypertension for 7 years, hypokalemia, plasma aldosterone/renin activity ratio greater than 40, normal adrenals in the CAT scan, and a negative genetic screening for GRA. She had normotensive pregnancies 5 and 3 years prior to the detection of hypertension, with hypertensive crisis in both postpartum periods, retrospectively considered as expressions of primary hyperaldosteronism.

  14. ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity1[OPEN

    PubMed Central

    Kimberlin, Athen N.; Chen, Ming; Dunn, Teresa M.

    2016-01-01

    Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network. PMID:27506241

  15. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    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.

  16. Aldosterone induces fibrosis, oxidative stress and DNA damage in livers of male rats independent of blood pressure changes

    SciTech Connect

    Queisser, Nina; Happ, Kathrin; Link, Samuel; Jahn, Daniel; Zimnol, Anna; Geier, Andreas; Schupp, Nicole

    2014-11-01

    Mineralocorticoid receptor blockers show antifibrotic potential in hepatic fibrosis. The mechanism of this protective effect is not known yet, although reactive oxygen species seem to play an important role. Here, we investigated the effects of elevated levels of aldosterone (Ald), the primary ligand of the mineralocorticoid receptor, on livers of rats in a hyperaldosteronism model: aldosterone-induced hypertension. Male Sprague–Dawley rats were treated for 4 weeks with aldosterone. To distinguish if damage caused in the liver depended on increased blood pressure or on increased Ald levels, the mineralocorticoid receptor antagonist spironolactone was given in a subtherapeutic dose, not normalizing blood pressure. To investigate the impact of oxidative stress, the antioxidant tempol was administered. Aldosterone induced fibrosis, detected histopathologically, and by expression analysis of the fibrosis marker, α-smooth muscle actin. Further, the mRNA amount of the profibrotic cytokine TGF-β was increased significantly. Fibrosis could be reduced by scavenging reactive oxygen species, and also by blocking the mineralocorticoid receptor. Furthermore, aldosterone treatment caused oxidative stress and DNA double strand breaks in livers, as well as the elevation of DNA repair activity. An increase of the transcription factor Nrf2, the main regulator of the antioxidative response could be observed, and of its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. All these effects of aldosterone were prevented by spironolactone and tempol. Already after 4 weeks of treatment, aldosteroneinfusion induced fibrosis in the liver. This effect was independent of elevated blood pressure. DNA damage caused by aldosterone might contribute to fibrosis progression when aldosterone is chronically increased. - Highlights: • Aldosterone has direct profibrotic effects on the liver independent of blood pressure. • Fibrosis is mediated by the mineralocorticoid receptor and

  17. Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats

    SciTech Connect

    Morales, Ana I.; Vicente-Sanchez, Cesar; Jerkic, Mirjana; Santiago, Jose M.; Sanchez-Gonzalez, Penelope D.; Perez-Barriocanal, Fernando; Lopez-Novoa, Jose M. . E-mail: jmlnovoa@usal.es

    2006-01-15

    Inflammation can play a key role in Cd-induced dysfunctions. Quercetin is a potent oxygen free radical scavenger and a metal chelator. Our aim was to study the effect of quercetin on Cd-induced kidney damage and metallothionein expression. The study was performed in Wistar rats that were administered during 9 weeks with either cadmium (1.2 mg Cd/kg/day, s.c.), quercetin (50 mg/kg/day, i.p.) or cadmium + quercetin. Renal toxicity was evaluated by measuring blood urea nitrogen concentration and urinary excretion of enzymes marker of tubular damage. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) renal expression were assessed by Western blot. Renal expression of metallothionein 1 and 2 (MT-1, MT-2) and eNOS mRNA was assessed by Northern blot. Our data demonstrated that Cd-induced renal toxicity was markedly reduced in rats that also received quercetin. MT-1 and MT-2 mRNA levels in kidney were substantially increased during treatment with Cd, being even higher when the animals received Cd and quercetin. Renal eNOS expression was significantly higher in rats receiving Cd and quercetin than in animals receiving Cd alone or in control rats. In the group that received Cd, COX-2 and iNOS expression was markedly higher than in control rats. In the group Cd + quercetin, no changes in COX-2 and iNOS expression were observed compared with the control group. Our results demonstrate that quercetin treatment prevents Cd-induced overexpression of iNOS and COX-2, and increases MT expression. These effects can explain the protection by quercetin of Cd-induced nephrotoxicity.

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

    SciTech Connect

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur; Nishimura, Kohji; Jisaka, Mitsuo; Nagaya, Tsutomu; Shono, Fumiaki; Yokota, Kazushige

    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

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

    PubMed

    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.

  20. Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

    PubMed

    Wang, Yechun; Miao, Zhiqi; Tang, Kexuan

    2010-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

  1. Suppression of Aldosterone Secretion After Recumbent Saline Infusion Does Not Exclude Lateralized Primary Aldosteronism.

    PubMed

    Cornu, Erika; Steichen, Olivier; Nogueira-Silva, Luis; Küpers, Elselien; Pagny, Jean-Yves; Grataloup, Christine; Baron, Stéphanie; Zinzindohoue, Franck; Plouin, Pierre-François; Amar, Laurence

    2016-10-01

    Guidelines recommend suppression tests such as the saline infusion test (SIT) to ascertain the diagnosis of primary aldosteronism (PA) in patients with a high aldosterone:renin ratio. However, suppression tests have only been evaluated in small retrospective series, and some experts consider that they are not helpful for the diagnosis of PA. In this study, we evaluated whether low post-SIT aldosterone concentrations do exclude lateralized PA. Between February 2009 and December 2013, 199 patients diagnosed with PA on the basis of 2 elevated aldosterone:renin ratio results and a high basal plasma or urinary aldosterone level or high post-SIT aldosterone level had a selective adrenal venous sampling. We used a selectivity index of 2 and a lateralization index of 4 to interpret the adrenal venous sampling results. Baseline characteristics of the patients were the following (percent or median): men 63%, 48 years old, office blood pressure 142/88 mm Hg, serum potassium 3.4 mmol/L, aldosterone:renin ratio 113 pmol/mU, plasma aldosterone concentration 588 pmol/L. The proportion of patients with lateralized adrenal venous sampling was 12 of 41 (29%) among those with post-SIT aldosterone <139 pmol/L (5 ng/dL) and 38 of 104 (37%) among those with post-SIT aldosterone <277 pmol/L (10 ng/dL). Post-SIT aldosterone levels were not associated with the blood pressure outcome of adrenalectomy. A low post-SIT aldosterone level cannot rule out lateralized PA, even with a low threshold (139 pmol/L). Adrenal venous sampling should be considered for patients who are eligible for surgery with elevated basal aldosterone levels even if they have low aldosterone concentrations after recumbent saline suppression testing.

  2. Construction of gene expression system in hop (Humulus lupulus) lupulin gland using valerophenone synthase promoter.

    PubMed

    Okada, Yukio; Saeki, Kazuo; Inaba, Akira; Suda, Narushi; Kaneko, Takafumi; Ito, Kazutoshi

    2003-09-01

    The promoter region of the valerophenone synthase (VPS) gene was isolated from hop (Humulus lupulus). VPS, a member of the chalcone synthase (CHS) super-family, catalyzes the biosynthesis reaction of the hop resin that significantly accumulates in the cone's secretory gland called the "lupulin gland". The typical H-box and G-box sequences, which exist in many plants' CHS promoters and act as cis-elements for tissue specificity, UV-light induction, etc., were not found in the isolated VPS promoter, although the H-box-like sequence (CCTTACC, CCTAACC) and the core sequence (ACGT) of the G-box were observed. The transformation experiment using the VPS promoter-UIDA gene fusion revealed that the promoter acts not only in the lupulin gland but also in the glands of leaf and stem. On the other hand, the VPS promoter activity was not induced by UV-irradiation.

  3. Ent-kaurene synthase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis.

    PubMed

    Kawaide, H; Imai, R; Sassa, T; Kamiya, Y

    1997-08-29

    ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10(-6) M, whereas it did not inhibit the ent-kaurene synthase activity even at 10(-4) M. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions.

  4. Geranyl diphosphate synthase from mint

    DOEpatents

    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.

  5. Geranyl diphosphate synthase from mint

    DOEpatents

    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.

  6. Imbalance in expression of hop (Humulus lupulus) chalcone synthase H1 and its regulators during hop stunt viroid pathogenesis.

    PubMed

    Füssy, Zoltán; Patzak, Josef; Stehlík, Jan; Matoušek, Jaroslav

    2013-05-01

    Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar "Admiral", including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

  7. Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

    PubMed

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-05

    Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.

  8. Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001.

    PubMed

    Nakai, T; Moriya, A; Tonouchi, N; Tsuchida, T; Yoshinaga, F; Horinouchi, S; Sone, Y; Mori, H; Sakai, F; Hayashi, T

    1998-06-15

    The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.

  9. Tissue- and development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase.

    PubMed Central

    Lee, M A; Cai, L; Hübner, N; Lee, Y A; Lindpaintner, K

    1997-01-01

    Nitric oxide (NO) functions as an intercellular messenger and mediates numerous biological functions. Among the three isoforms of NO synthase that produce NO, the ubiquitously expressed neuronal NO synthase (nNOS) is responsible for a large part of NO production, yet its regulation is poorly understood. Recent reports of two alternative spliceforms of nNOS in the mouse and in man have raised the possibility of spatial and temporal modulation of expression. This study demonstrates the existence of at least three transcripts of the rat nNOS gene designated nNOSa, nNOSb, and nNOSc, respectively, with distinct 5' untranslated first exons that arise from alternative splicing to a common second exon. Expression of the alternative transcripts occurs with a high degree of tissue and developmental specificity, as demonstrated by RNase protection assays on multiple tissues from both fetal and adult rats. Furthermore, terminal differentiation of rat pheochromocytoma-derived PC12 cells into neurons is associated with induction of nNOSa, suggesting, likewise, development- and tissue-specific transcriptional control of nNOS isoform expression. Physical mapping using a rat yeast artificial chromosome clone shows that the alternatively spliced first exons 1a, 1b, and 1c are separated by at least 15-60 kb from the downstream coding sequence, with exons 1b and 1c being positioned within 200 bp of each other. These findings provide evidence that the biological activity of nNOS is tightly and specifically regulated by a complex pattern of alternative splicing, indicating that the notion of constitutive expression of this isoform needs to be revised. PMID:9294118

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

    PubMed Central

    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. PMID:15973775

  11. In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken Sertoli cells.

    PubMed

    Hassanpour, H; Mirshokraei, P; Sadrabad, E Khalili; Dehkordi, A Esmailian; Layeghi, S; Afzali, A; Mohebbi, A

    2015-02-01

    To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes.

  12. One type of chalcone synthase gene expressed during embryogenesis regulates the flavonoid accumulation in citrus cell cultures.

    PubMed

    Moriguchi, T; Kita, M; Tomono, Y; EndoInagaki, T; Omura, M

    1999-06-01

    To elucidate the relationship between the expression of chalcone synthase (CHS) genes and the production of flavonoid in citrus cell cultures, two cDNA clones encoding CHS were isolated (CitCHS1 and CitCHS2) from the citrus. The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not. There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids. These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoid in citrus cell cultures.

  13. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    PubMed

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue.

  14. Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase.

    PubMed

    Neily, Mohamed Hichem; Matsukura, Chiaki; Maucourt, Mickaël; Bernillon, Stéphane; Deborde, Catherine; Moing, Annick; Yin, Yong-Gen; Saito, Takeshi; Mori, Kentaro; Asamizu, Erika; Rolin, Dominique; Moriguchi, Takaya; Ezura, Hiroshi

    2011-02-15

    Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.

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

    SciTech Connect

    Huang, T.-Y.; Chu, H.-C.; Lin, Y.-L.; Lin, C.-K.; Hsieh, T.-Y.; Chang, W.-K.; Chao, Y.-C.; Liao, C.-L.

    2009-05-15

    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.

  16. Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

    PubMed

    Erez-Roman, Racheli; Pienik, Reut; Futerman, Anthony H

    2010-01-01

    Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

  17. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  18. Bioinformatic comparisons and tissue expression of the neuronal nitric oxide synthase (nNOS) gene from the red drum (Sciaenops ocellatus).

    PubMed

    Zhou, Libin; Bai, Ru; Tian, Jianxiao; Liu, Xiaochun; Lu, Danqi; Zhu, Pei; Liu, Yun; Zeng, Lujiao; Luo, Wenna; Zhang, Yong; Wang, Anli

    2009-10-01

    The full length cDNA sequence for neuronal nitric oxide synthase (nNOS) gene from red drum (Sciaenops ocellatus) has been cloned, subjected to bioinformatic analysis, and examined for expression in different tissues. Red drum nNOS showed high identity to nNOS of mammals and other fish species. Notably, a unique 7-aa insertion was found in the important catalytic sites of the NO synthase domain, possibly affecting the function of red drum nNOS. Furthermore, this nNOS was expressed not only in brain but also in most of the internal organs including liver, intestine, spleen, head kidney and thymus.

  19. Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis.

    PubMed

    Chen, X Y; Chen, Y; Heinstein, P; Davisson, V J

    1995-12-20

    In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. A Gossypium arboreum cell suspension culture which produces the sesquiterpene phytoalexin gossypol showed a time-dependent 10-fold increase in a 1.9-kb mRNA in response to a challenge by a preparation from Verticillium dahliae. The mRNA prepared from these elicited cultures was used to isolated two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 and 64,118. The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases. Western blot analyses with a cross-reactive monoclonal antibody from a related sesquiterpene synthase in Nicotiana tabacum showed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor treatment. The encoded protein from the pXC1 cDNA was produced in Escherichia coli and purified by affinity column chromatography. The enzymatic properties of this protein were identified by a radiochemical assay for cyclization of farnesyldiphosphate and a product structure was assigned by GC-MS, chiral phase GC, and NMR analyses as (+)-delta-cadinene. The fungal-elicited production of a (+)-delta-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton.

  20. Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa.

    PubMed

    Barone, Pierluigi; Rosellini, Daniele; Lafayette, Peter; Bouton, Joseph; Veronesi, Fabio; Parrott, Wayne

    2008-05-01

    Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants were assayed for exclusion of Al from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Overall, only the soil assays yielded consistent results. Based on the soil assays, two transgenic events were identified that were more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance.

  1. Cloning, expression, and characterization of para-aminobenzoic acid (PABA) synthase from Agaricus bisporus 02, a thermotolerant mushroom strain.

    PubMed

    Deng, Li-Xin; Shen, Yue-Mao; Song, Si-Yang

    2015-01-01

    The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.

  2. Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis

    PubMed Central

    Pazouki, Leila; Memari, Hamid R.; Kännaste, Astrid; Bichele, Rudolf; Niinemets, Ülo

    2015-01-01

    Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes. PMID:25784918

  3. Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis.

    PubMed

    Pazouki, Leila; Memari, Hamid R; Kännaste, Astrid; Bichele, Rudolf; Niinemets, Ülo

    2015-01-01

    Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  4. Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

    PubMed

    Jokela, Tiina; Kärnä, Riikka; Rauhala, Leena; Bart, Genevieve; Pasonen-Seppänen, Sanna; Oikari, Sanna; Tammi, Markku I; Tammi, Raija H

    2017-03-24

    The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

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

    SciTech Connect

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    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)

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

    PubMed

    Ozbek, Emin; Simsek, Abdulmuttalip; Ozbek, Mustafa; Somay, Adnan

    2013-09-26

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

    PubMed

    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.

  8. Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin.

    PubMed

    Sidgwick, Gary P; Iqbal, Syed A; Bayat, Ardeshir

    2013-05-01

    Keloid disease (KD) is a fibroproliferative disorder characterised partly by an altered extracellular matrix (ECM) profile. In fetal scarring, hyaluronic acid (HA) expression is increased, but is reduced in KD tissue compared with normal skin (NS). The expression of Hyaluronan Synthase (HAS) and hyaluronidase (HYAL) in KD and NS tissue were investigated for the first time using a range of techniques. Hyaluronan synthase and HYAL mRNA expression were significantly increased in NS tissue compared with KD tissue (P < 0.05). Immunohistological analysis of tissue indicated an accumulation of HAS and HYAL protein expression in KD compared with NS due to the thicker epidermis. No differences were observed in mRNA or protein expression in KD and NS fibroblasts. Reduced expression of HAS and HYAL may alter HA synthesis, degradation and accumulation in KD. Better understanding of the role of HA in KD may lead to novel therapeutic approaches to address the resulting ECM imbalance.

  9. Reverse remodeling and recovery from cachexia in rats with aldosteronism.

    PubMed

    Cheema, Yaser; Zhao, Wenyuan; Zhao, Tieqiang; Khan, M Usman; Green, Kelly D; Ahokas, Robert A; Gerling, Ivan C; Bhattacharya, Syamal K; Weber, Karl T

    2012-08-15

    The congestive heart failure (CHF) syndrome with soft tissue wasting, or cachexia, has its pathophysiologic origins rooted in neurohormonal activation. Mechanical cardiocirculatory assistance reveals the potential for reverse remodeling and recovery from CHF, which has been attributed to device-based hemodynamic unloading whereas the influence of hormonal withdrawal remains uncertain. This study addresses the signaling pathways induced by chronic aldosteronism in normal heart and skeletal muscle at organ, cellular/subcellular, and molecular levels, together with their potential for recovery (Recov) after its withdrawal. Eight-week-old male Sprague-Dawley rats were examined at 4 wk of aldosterone/salt treatment (ALDOST) and following 4-wk Recov. Compared with untreated, age-/sex-/strain-matched controls, ALDOST was accompanied by 1) a failure to gain weight, reduced muscle mass with atrophy, and a heterogeneity in cardiomyocyte size across the ventricles, including hypertrophy and atrophy at sites of microscopic scarring; 2) increased cardiomyocyte and mitochondrial free Ca(2+), coupled to oxidative stress with increased H(2)O(2) production and 8-isoprostane content, and increased opening potential of the mitochondrial permeability transition pore; 3) differentially expressed genes reflecting proinflammatory myocardial and catabolic muscle phenotypes; and 4) reversal to or toward recovery of these responses with 4-wk Recov. Aldosteronism in rats is accompanied by cachexia and leads to an adverse remodeling of the heart and skeletal muscle at organ, cellular/subcellular, and molecular levels. However, evidence presented herein implicates that these tissues retain their inherent potential for recovery after complete hormone withdrawal.

  10. SFE/SFHTA/AFCE primary aldosteronism consensus: Introduction and handbook.

    PubMed

    Amar, Laurence; Baguet, Jean Philippe; Bardet, Stéphane; Chaffanjon, Philippe; Chamontin, Bernard; Douillard, Claire; Durieux, Pierre; Girerd, Xaxier; Gosse, Philippe; Hernigou, Anne; Herpin, Daniel; Houillier, Pascal; Jeunemaitre, Xavier; Joffre, Francis; Kraimps, Jean-Louis; Lefebvre, Hervé; Ménégaux, Fabrice; Mounier-Véhier, Claire; Nussberger, Juerg; Pagny, Jean-Yves; Pechère, Antoinette; Plouin, Pierre-François; Reznik, Yves; Steichen, Olivier; Tabarin, Antoine; Zennaro, Maria-Christina; Zinzindohoue, Franck; Chabre, Olivier

    2016-07-01

    The French Endocrinology Society (SFE) French Hypertension Society (SFHTA) and Francophone Endocrine Surgery Association (AFCE) have drawn up recommendations for the management of primary aldosteronism (PA), based on an analysis of the literature by 27 experts in 7 work-groups. PA is suspected in case of hypertension associated with one of the following characteristics: severity, resistance, associated hypokalemia, disproportionate target organ lesions, or adrenal incidentaloma with hypertension or hypokalemia. Diagnosis is founded on aldosterone/renin ratio (ARR) measured under standardized conditions. Diagnostic thresholds are expressed according to the measurement units employed. Diagnosis is established for suprathreshold ARR associated with aldosterone concentrations >550pmol/L (200pg/mL) on 2 measurements, and rejected for aldosterone concentration<240pmol/L (90pg/mL) and/or subthreshold ARR. The diagnostic threshold applied is different if certain medication cannot be interrupted. In intermediate situations, dynamic testing is performed. Genetic forms of PA are screened for in young subjects and/or in case of familial history. The patient should be informed of the results expected from medical and surgical treatment of PA before exploration for lateralization is proposed. Lateralization is explored by adrenal vein sampling (AVS), except in patients under 35 years of age with unilateral adenoma on imaging. If PA proves to be lateralized, unilateral adrenalectomy may be performed, with adaptation of medical treatment pre- and postoperatively. If PA is non-lateralized or the patient refuses surgery, spironolactone is administered as first-line treatment, replaced by amiloride, eplerenone or calcium-channel blockers if insufficiently effective or poorly tolerated.

  11. The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

    PubMed

    Bousquet, Elodie; Zhao, Min; Ly, André; Leroux Les Jardins, Guillaume; Goldenberg, Brigitte; Naud, Marie-Christine; Jonet, Laurent; Besson-Lescure, Bernadette; Jaisser, Frederic; Farman, Nicolette; De Kozak, Yvonne; Behar-Cohen, Francine

    2012-01-01

    We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

  12. In vivo Expression of Inducible Nitric Oxide Synthase in Experimentally Induced Neurologic Diseases

    NASA Astrophysics Data System (ADS)

    Koprowski, Hilary; Zheng, Yong Mu; Heber-Katz, Ellen; Fraser, Nigel; Rorke, Lucy; Fu, Zhen Fang; Hanlon, Cathleen; Dietzschold, Bernhard

    1993-04-01

    The purpose of this study was to investigate the induction of inducible nitric oxide synthase (iNOS) mRNA in the brain tissue of rats and mice under the following experimental conditions: in rats infected with borna disease virus and rabies virus, in mice infected with herpes simplex virus, and in rats after the induction of experimental allergic encephalitis. The results showed that iNOS mRNA, normally nondetectable in the brain, was present in animals after viral infection or after induction of experimental allergic encephalitis. The induction of iNOS mRNA coincided with the severity of clinical signs and in some cases with the presence of inflammatory cells in the brain. The results indicate that nitric oxide produced by cells induced by iNOS may be the toxic factor accounting for cell damage and this may open the door to approaches to the study of the pathogenesis of neurological diseases.

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

    PubMed Central

    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

  14. Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.

    PubMed

    Adams, S H; Alho, C S; Asins, G; Hegardt, F G; Marrero, P F

    1997-05-15

    The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.

  15. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.

    PubMed

    Muñoz-Bertomeu, Jesús; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2008-01-01

    We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

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

    PubMed

    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.

  17. WdCHS3, a Gene That Encodes a Class III Chitin Synthase in Wangiella (Exophiala) dermatitidis, Is Expressed Differentially under Stress Conditions

    PubMed Central

    Wang, Zheng; Szaniszlo, Paul J.

    2000-01-01

    Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5′ upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between −780 and −1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5′ upstream sequence. PMID:10648509

  18. Fatty acid synthase 2 contributes to diapause preparation in a beetle by regulating lipid accumulation and stress tolerance genes expression

    PubMed Central

    Tan, Qian-Qian; Liu, Wen; Zhu, Fen; Lei, Chao-Liang; Wang, Xiao-Ping

    2017-01-01

    Diapause, also known as dormancy, is a state of arrested development that allows insects to survive unfavorable environmental conditions. Diapause-destined insects store large amounts of fat when preparing for diapause. However, the extent to which these accumulated fat reserves influence diapause remains unclear. To address this question, we investigated the function of fatty acid synthase (FAS), which plays a central role in lipid synthesis, in stress tolerance, the duration of diapause preparation, and whether insects enter diapause or not. In diapause-destined adult female cabbage beetles, Colaphellus bowringi, FAS2 was more highly expressed than FAS1 at the peak stage of diapause preparation. FAS2 knockdown suppressed lipid accumulation and subsequently affected stress tolerance genes expression and water content. However, silencing FAS2 had no significant effects on the duration of diapause preparation or the incidence of diapause. FAS2 transcription was suppressed by juvenile hormone (JH) and the JH receptor methoprene-tolerant (Met). These results suggest that the absence of JH-Met induces FAS2 expression, thereby promoting lipid storage in diapause-destined female beetles. These results demonstrate that fat reserves regulate stress tolerance genes expression and water content, but have no significant effect on the duration of diapause preparation or the incidence of diapause. PMID:28071706

  19. Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

    PubMed Central

    Kadono, Takashi; Kira, Nozomu; Suzuki, Kengo; Iwata, Osamu; Ohama, Takeshi; Okada, Shigeru; Nishimura, Tomohiro; Akakabe, Mai; Tsuda, Masashi; Adachi, Masao

    2015-01-01

    Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase. PMID:26308005

  20. TCP transcription factors are critical for the coordinated regulation of isochorismate synthase 1 expression in Arabidopsis thaliana.

    PubMed

    Wang, Xiaoyan; Gao, Jiong; Zhu, Zheng; Dong, Xianxin; Wang, Xiaolei; Ren, Guodong; Zhou, Xin; Kuai, Benke

    2015-04-01

    Salicylic acid (SA) plays an important role in various aspects of plant development and responses to stresses. To elucidate the sophisticated regulatory mechanism of SA synthesis and signaling, we used a yeast one-hybrid system to screen for regulators of isochorismate synthase 1 (ICS1), a gene encoding the key enzyme in SA biosynthesis in Arabidopsis thaliana. A TCP family transcription factor AtTCP8 was initially identified as a candidate regulator of ICS1. The regulation of ICS1 by TCP proteins is supported by the presence of a typical TCP binding site in the ICS1 promoter. The binding of TCP8 to this site was confirmed by in vitro and in vivo assays. Expression patterns of TCP8 and its corresponding gene TCP9 largely overlapped with ICS1 under pathogen attack. A significant reduction in the expression of ICS1 during immune responses was observed in the tcp8 tcp9 double mutant. We also detected strong interactions between TCP8 and SAR deficient 1 (SARD1), WRKY family transcription factor 28 (WRKY28), NAC (NAM/ATAF1,ATAF2/CUC2) family transcription factor 019 (NAC019), as well as among TCP8, TCP9 and TCP20, suggesting a complex coordinated regulatory mechanism underlying ICS1 expression. Our results collectively demonstrate that TCP proteins are involved in the orchestrated regulation of ICS1 expression, with TCP8 and TCP9 being verified as major representatives.

  1. Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression.

    PubMed

    Satoh, A; Shimosegawa, T; Kimura, K; Moriizumi, S; Masamune, A; Koizumi, M; Toyota, T

    1998-11-01

    To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.

  2. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

    PubMed Central

    Majano, P L; García-Monzón, C; López-Cabrera, M; Lara-Pezzi, E; Fernández-Ruiz, E; García-Iglesias, C; Borque, M J; Moreno-Otero, R

    1998-01-01

    Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis. PMID:9525976

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

    PubMed

    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.

  4. Body mass index and risk of colorectal cancer according to fatty acid synthase expression in the nurses' health study.

    PubMed

    Kuchiba, Aya; Morikawa, Teppei; Yamauchi, Mai; Imamura, Yu; Liao, Xiaoyun; Chan, Andrew T; Meyerhardt, Jeffrey A; Giovannucci, Edward; Fuchs, Charles S; Ogino, Shuji

    2012-03-07

    Fatty acid synthase (FASN) plays an important role in energy metabolism of fatty acids and is overexpressed in some colon cancers. We investigated whether associations between body mass index (BMI) and risk of colorectal cancer varied according to FASN expression. During follow-up of 109,051 women in the ongoing prospective Nurses' Health Study, a total of 1351 incident colon and rectal cancers were diagnosed between 1986 and 2004. We constructed tissue microarrays of the available resected tumor samples (n = 536), and FASN expression was analyzed by immunohistochemistry. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazards regression models. All statistical tests were two-sided. High BMI was associated with an increased risk of FASN-negative (no or weak expression) colorectal cancer compared with normal BMI (high BMI [≥ 30 kg/m(2)], ie, obese vs normal BMI [18.5-22.9 kg/m(2)], HR = 2.25, 95% CI = 1.49 to 3.40, P(trend) < .001) but not with FASN-positive (moderate to strong expression) colorectal cancer. A statistically significant heterogeneity in colorectal cancer risks was observed between FASN-negative and FASN-positive tumors (P(heterogeneity) = .033). The age-adjusted incidence rates for FASN-positive and FASN-negative colorectal cancers were 10.9 and 7.1, respectively, per 100,000 person-years. This molecular pathological epidemiology study supports a role of energy metabolism in colorectal cancer pathogenesis.

  5. Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure

    PubMed Central

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-01-01

    This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS. PMID:27191892

  6. Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

    PubMed

    Hanaoka, Ryuki; Dawid, Igor B; Kawahara, Atsuo

    2007-02-01

    Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.

  7. The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed.

    PubMed Central

    Ruan, Y. L.; Chourey, P. S.; Delmer, D. P.; Perez-Grau, L.

    1997-01-01

    Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism. PMID:12223814

  8. Aldosterone Inactivates the Endothelin-B Receptor via a Cysteinyl Thiol Redox Switch to Decrease Pulmonary Endothelial Nitric Oxide Levels and Modulate Pulmonary Arterial Hypertension

    PubMed Central

    Maron, Bradley A.; Zhang, Ying-Yi; White, Kevin; Chan, Stephen Y.; Handy, Diane E.; Mahoney, Christopher E.; Loscalzo, Joseph; Leopold, Jane A.

    2012-01-01

    Background Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO•) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ETB), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species (ROS) generation and decreasing NO• levels. We hypothesized that aldosterone modulates PAH by disrupting ETB-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. Methods and Results In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO• metabolites in the absence of left heart failure. In human pulmonary artery endothelial cells (HPAECs), endothelin-1 increased aldosterone levels via PGC-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased ROS production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ETB to decrease endothelin-1-stimulated eNOS activity. Substitution of ETB-Cys405 with alanine improved ETB-dependent NO• synthesis under conditions of oxidant stress, confirming that Cys405 is a redox sensitive thiol that is necessary for ETB-eNOS signaling. In HPAECs, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated ROS generation and restored ETB-dependent NO• production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in two animal models of PAH in vivo. Conclusions Our findings demonstrate that aldosterone modulates an ETB cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO• and promote PAH

  9. Expression of the naphthoate synthase gene in Mycobacterium tuberculosis in a self-generated oxygen depleted liquid culture system.

    PubMed

    Ramchandra, Prathna; Sturm, A Willem

    2010-12-01

    Mycobacterium tuberculosis has been classified for decades as a strict aerobic species. Whole genome sequencing of the type culture strain H37Rv has revealed the presence of a full set of genes allowing for anaerobic metabolism. Naphthoate synthase (menB) is a key enzyme required for the synthesis of menaquinone, which plays a crucial role in anaerobic electron transport, ultimately resulting in the formation of energy generating intermediates. Interrupting the synthesis of this enzyme will interfere with the production of menaquinone and therefore this enzyme is a potential drug target. This study serves to investigate the role of naphtoate synthase in the survival of M. tuberculosis H37Rv when incubated under oxygen limiting conditions of unagitated liquid culture over 15 weeks. M. tuberculosis H37Rv was grown in Middlebrook 7H9 media. The tubes were kept undisturbed at 37 °C for up to 15 weeks. At selected time points, aliquots of cells were removed and frozen. RNA was simultaneously extracted from all aliquots. The RNA was converted to cDNA for Real-Time PCR on the ABI 7000 SDS. Gene expression was normalized against 16S RNA quantities at each time point. A systematic increase in the expression of the menB gene product was observed over the incubation period with a 4.3-fold increase seen at week 6 (P < 0.001) relative to day 0 and an 85.8-fold increase at week 15 (P < 0.001) relative to day 0. Cells of M. tuberculosis increase menaquinone production during prolonged incubation in broth culture as a mechanism of survival. This study substantiates the menB enzyme to be a putative drug target.

  10. Molecular cloning and differential expression analysis of a squalene synthase gene from Dioscorea zingiberensis, an important pharmaceutical plant.

    PubMed

    Ye, Yun; Wang, Runfa; Jin, Liang; Shen, Junhao; Li, Xiaotong; Yang, Ting; Zhou, Mengzhuo; Yang, Zhifan; Chen, Yongqin

    2014-09-01

    Diosgenin is a steroid derived from cholesterol in plants and used as a typical initial intermediate for synthesis of numerous steroidal drugs in the world. Commercially, this compound is extracted mainly from the rhizomes or tubers of some Dioscorea species. Squalene synthase (SQS: EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene, the first committed step for biosynthesis of plant sterols including cholesterol, and is thought to play an important role in diosgenin biosynthesis. A full-length cDNA of a putative squalene synthase gene was cloned from D. zingiberensis and designated as DzSQS (Genbank Accession Number KC960673). DzSQS was contained an open reading frame of 1,230 bp encoding a polypeptide of 409 amino acids with a predicted molecular weight of 46 kDa and an isoelectric point of 6.2. The deduced amino acid sequence of DzSQS shared over 70 % sequence identity with those of SQSs from other plants. The truncated DzSQS in which 24 amino acids were deleted from the carboxy terminus was expressed in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. Quantitative real-time PCR analysis revealed that DzSQS was expressed from highest to lowest order in mature leaves, newly-formed rhizomes, young leaves, young stems, and two-year-old rhizomes of D. zingiberensis.

  11. Primary Aldosteronism and ARMC5 Variants

    PubMed Central

    Zilbermint, Mihail; Xekouki, Paraskevi; Faucz, Fabio R.; Berthon, Annabel; Gkourogianni, Alexandra; Schernthaner-Reiter, Marie Helene; Batsis, Maria; Sinaii, Ninet; Quezado, Martha M.; Merino, Maria; Hodes, Aaron; Abraham, Smita B.; Libé, Rossella; Assié, Guillaume; Espiard, Stéphanie; Drougat, Ludivine; Ragazzon, Bruno; Davis, Adam; Gebreab, Samson Y.; Neff, Ryan; Kebebew, Electron; Bertherat, Jérôme; Lodish, Maya B.

    2015-01-01

    Context: Primary aldosteronism is one of the leading causes of secondary hypertension, causing significant morbidity and mortality. A number of genetic defects have recently been identified in primary aldosteronism, whereas we identified mutations in ARMC5, a tumor-suppressor gene, in cortisol-producing primary macronodular adrenal hyperplasia. Objective: We investigated a cohort of 56 patients who were referred to the National Institutes of Health for evaluation of primary aldosteronism for ARMC5 defects. Methods: Patients underwent step-wise diagnosis, with measurement of serum aldosterone and plasma renin activity followed by imaging, saline suppression and/or oral salt loading tests, plus adrenal venous sampling. Cortisol secretion was also evaluated; unilateral or bilateral adrenalectomy was performed, if indicated. DNA, protein, and transfection studies in H295R cells were conducted by standard methods. Results: We identified 12 germline ARMC5 genetic alterations in 20 unrelated and two related individuals in our cohort (39.3%). ARMC5 sequence changes in 6 patients (10.7%) were predicted to be damaging by in silico analysis. All affected patients carrying a variant predicted to be damaging were African Americans (P = .0023). Conclusions: Germline ARMC5 variants may be associated with primary aldosteronism. Additional cohorts of patients with primary aldosteronism and metabolic syndrome, particularly African Americans, should be screened for ARMC5 sequence variants because these may underlie part of the known increased predisposition of African Americans to low renin hypertension. PMID:25822102

  12. Relaxin exerts two opposite effects on mechanical activity and nitric oxide synthase expression in the mouse colon.

    PubMed

    Baccari, M C; Traini, C; Garella, R; Cipriani, G; Vannucchi, M G

    2012-11-01

    The hormone relaxin exerts a variety of functions on the smooth muscle of reproductive and nonreproductive organs, most of which occur through a nitric oxide (NO)-mediated mechanism. In the stomach and ileum, relaxin causes muscle relaxation by modulating the activity and expression of different nitric oxide synthase (NOS) isoforms region-dependently. Nothing is known on the effects of relaxin in the colon, the gut region expressing the highest number of neuronal (n) NOSβ-immunoreactive neurons and mainly involved in motor symptoms of pregnancy and menstrual cycle. Therefore, we studied the effects of relaxin exposure in the mouse proximal colon in vitro evaluating muscle mechanical activity and NOS isoform expression. The functional experiments showed that relaxin decreases muscle tone and increases amplitude of spontaneous contractions; the immunohistochemical results showed that relaxin increases nNOSβ and endothelial (e) NOS expression in the neurons and decreases nNOSα and eNOS expression in the smooth muscle cells (SMC). We hypothesized that, in the colon, relaxin primarily increases the activity and expression of nNOSβ and eNOS in the neurons, causing a reduction of the muscle tone. The downregulation of nNOSα and eNOS expression in the SMC associated with increased muscle contractility could be the consequence of continuous exposue of these cells to the NO of neuronal origin. These findings may help to better understand the physiology of NO in the gastrointestinal tract and the role that the "relaxin-NO" system plays in motor disorders such as functional bowel disease.

  13. Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress.

    PubMed

    Lu, Shanfa; Li, Laigeng; Yi, Xiaoping; Joshi, Chandrashekhar P; Chiang, Vincent L

    2008-01-01

    Trees constitute the majority of lignocellulosic biomass existing on our planet. Trees also serve as important feedstock materials for various industrial products. However, little is known about the regulatory mechanisms of cellulose synthase (CesA) genes of trees. Here, the cloning and characterization of three CesA genes (EgraCesA1, EgraCesA2, and EgraCesA3) from an economically important tree species, Eucalyptus grandis, are reported. All three genes were specifically expressed in xylem cells of eucalyptus undergoing secondary cell wall biosynthesis. The GUS gene, expressed under the control of the EgraCesA2 or EgraCesA3 promoter, was also localized in the secondary xylem in transgenic tobacco stems. However, the EgraCesA1 promoter alone or along with its 5'-UTR introns was insufficient to direct appropriate GUS expression. EgraCesA2 and EgraCesA3 gene expression was up-regulated in tension-stressed eucalyptus xylem cells. Accordingly, GUS expression directed by the EgraCesA2 or EgraCesA3 promoter was also up-regulated. EgraCesA1 had no such response. Thus, it is most unlikely that EgraCesA1 is a subunit of the EgraCesA2-EgraCesA3 complex. The presence of at least two types of cellulose biosynthesis machinery in wood formation is an important clue in deciphering the underpinnings of the perennial growth of trees in various environmental conditions. By analysing GUS gene expression directed by the EgraCesA3 promoter or its deletions, several negative and positive regulatory regions controlling gene expression in xylem or phloem were identified. Also a region which is likely to contain mechanical stress-responsive elements was deduced. These results will guide further studies on identifying cis-regulatory elements directing CesA gene transcription and wood formation regulatory networks.

  14. Chondroitin sulfate synthase 1 expression is associated with malignant potential of soft tissue sarcomas with myxoid substance.

    PubMed

    Momose, Takashige; Yoshimura, Yasuo; Harumiya, Satoru; Isobe, Ken'ichi; Kito, Munehisa; Fukushima, Mana; Kato, Hiroyuki; Nakayama, Jun

    2016-04-01

    The glycosyltransferases chondroitin sulfate synthase 1 (CHSY1) and exostoses-like 3 (EXTL3) specifically function in biosynthesis of the glycans chondroitin sulfate and heparan sulfate, respectively. Although these glycans play important roles in pathogenesis of various tumors, their significance in soft tissue sarcoma remains unknown. Here, we asked whether CHSY1 or EXTL3 expression correlates with malignant potential of soft tissue sarcomas with myxoid substance. To do so, we examined 40 samples representing specific types, including 12 cases of myxoid liposarcoma, 14 of myxofibrosarcoma, 12 of malignant peripheral nerve sheath tumor, and 2 of low-grade fibromyxoid sarcoma. We performed immunohistochemistry with anti-CHSY1 and anti-EXTL3 antibodies and compared enzyme expression levels with tumor histologic grade as assessed by the Fédération Nationale des Centres de Lutte Contre le Cancer classification and with patient 5-year survival rate. CHSY1 and EXTL3 were expressed in 72.5% and 32.5% of all tumors, respectively. Notably, CHSY1 was strongly expressed in myxofibrosarcoma and malignant peripheral nerve sheath tumor compared with other tumors and significantly associated with higher- rather than lower-grade tumors (P < .01). High expression of CHSY1 was also significantly associated with poorer patient outcomes (P = .031) and higher stages assessed by American Joint Committee on Cancer staging system (P = .004). By contrast, EXTL3 expression was not correlated with histologic grade or patient prognosis. We conclude that CHSY1 expression is closely associated with malignant potential of soft tissue sarcomas with myxoid substance.

  15. Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation.

    PubMed

    Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj

    2016-10-11

    Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation.

  16. Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation

    PubMed Central

    Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj

    2016-01-01

    Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation. PMID:27725707

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

    PubMed Central

    2011-01-01

    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 and pollen

  18. Excess aldosterone is a critical danger signal for inflammasome activation in the development of renal fibrosis in mice.

    PubMed

    Kadoya, Hiroyuki; Satoh, Minoru; Sasaki, Tamaki; Taniguchi, Shun'ichiro; Takahashi, Masafumi; Kashihara, Naoki

    2015-09-01

    High levels of aldosterone impair renal function by activating proinflammatory and profibrotic pathways. However, the molecular mechanism underlying aldosterone-induced inflammation and fibrosis is unknown. Inflammasome activation contributes to chronic kidney disease. We hypothesized that aldosterone induces renal tubulointerstitial inflammation and fibrosis by activating the inflammasome. Infusing wild-type mice with aldosterone (0.25 mg/kg/d) caused tubulointerstitial damage, increased expression of inflammasome components, caspase 1 activation, and overproduction of IL-1β and IL-18. These changes were suppressed by eplerenone treatment (100 mg/kg/d) in wild-type mice or in mice deficient in apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC). Caspase 1-positive and F4/80-positive cells colocalized in the interstitium. Bone marrow transplantation using ASC-deficient mice indicated that inflammasome activation in macrophages mediated aldosterone-induced renal fibrosis. IL-18 was detected in culture supernatants of macrophages treated with aldosterone, and mitochondria-derived reactive oxygen species activated the inflammasome in these macrophages. Our results indicate that exposure of macrophages to high levels of aldosterone resulted in the activation of inflammasomes via the mitochondria-derived reactive oxygen species. Thus, inflammasome activation in macrophages may serve as a new therapeutic target for chronic kidney disease.

  19. Circadian Rhythm of Plasma Aldosterone Concentration in Patients with Primary Aldosteronism

    PubMed Central

    Kem, David C.; Weinberger, Myron H.; Gomez-Sanchez, Celso; Kramer, Norman J.; Lerman, Robert; Furuyama, Shunsuke; Nugent, Charles A.

    1973-01-01

    Plasma aldosterone, cortisol, and renin activity were measured in nine recumbent patients with hyperaldosteronism, including seven with adenomas, one with idiopathic hyperplasia, and one with glucocorticoid suppressible hyperplasia. All had peak values of plasma aldosterone concentration from 3 a.m. to noon and lowest values at 6 p.m. or midnight. This rhythm was similar to the circadian pattern of plasma cortisol in the same patients. When these data were normalized to eliminate the wide variation in ranges of plasma aldosterone and cortisol between individuals, there was an excellent correlation (r = + 0.87, P < 0.005) between the two hormones. In contrast, plasma aldosterone concentrations did not correlate with plasma renin activity before or after normalization of data. Short term suppression of ACTH by administration of dexamethasone eliminated the circadian variation of plasma aldosterone in both patients with hyperplasia and in four of five patients with adenomas, while it markedly altered the rhythm in the fifth. Similar doses of dexamethasone were administered to four normal subjects and did not flatten the circadian rhythm of plasma aldosterone. These data suggest that patients with primary aldosteronism have a circadian rhythm of plasma aldosterone mediated by changes in ACTH. PMID:4353776

  20. Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers.

    PubMed

    Wen, Xiao-Peng; Pang, Xiao-Ming; Matsuda, Narumi; Kita, Masayuki; Inoue, Hiromichi; Hao, Yu-Jin; Honda, Chikako; Moriguchi, Takaya

    2008-04-01

    An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

  1. Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition.

    PubMed

    Welsh, Toni; Mitchell, Carolyn M; Walters, William A; Mesiano, Sam; Zakar, Tamas

    2005-12-15

    Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour.

  2. Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii

    PubMed Central

    Modarresi, Farzan; Azizi, Omid; Shakibaie, Mohammad Reza; Motamedifar, Mohammad; Mansouri, Shahla

    2016-01-01

    Background and Objectives: In present study we aimed to clone the luxI gene encoding N-acyl-homoserine synthase detected in clinical isolates of Acinetobacter baumannii and study its expression in Escherichia coli transformants. Materials and Methods: Four A. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. The presence of luxI gene was detected using PCR technique. Purified PCR product DNA was initially cloned into pTG19 and transformed to E. coli DH5α. The gene was then recovered from agarose gel and ligated by T4 DNA ligase into pET28a expression vector using NdeI and XhoI enzymes. pET28a + luxI was transformed into E. coli BL21 (DE3). The luxI putative gene was further detected in the transformants by colony PCR. Expression of the luxI gene in the recombinant E. coli BL21 cells was studied by quantitative real time PCR (qRT-PCR) and the presence of N-acylhomoserine lactone (AHL) was checked by colorimetric assay and Fourier Transform Infra-Red (FT-IR) spectroscopy. Results: We successfully cloned AHL gene from A. baumannii strain 23 to pET28a expression vector. There was four fold increases in expression of luxI in the transformants (P ≤ 0.05). It was found that, strain 23 and the transformants showed highest amount of AHL activity (OD = 1.524). The FT-IR analysis indicated stretching C=O bond of the lactone ring and primary amides (N=H) at 1764.69 cm−1 and 1659.23 cm−1 respectively. Conclusion: From above results we concluded that, luxI in A. baumannii is indeed responsible for AHL production and not regulation and pET28a vector allows efficient AHL expression in E. coli BL21 transformants. PMID:27307980

  3. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

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

    PubMed

    Hua-Huy, Thông; Duong-Quy, Sy; Pham, Hoa; Pansiot, Julien; Mercier, Jean-Christophe; Baud, Olivier; Dinh-Xuan, Anh Tuan

    2016-01-01

    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.

  5. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures.

    PubMed

    Dawson, V L; Brahmbhatt, H P; Mong, J A; Dawson, T M

    1994-11-01

    Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

  6. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    PubMed

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

  7. Molecular cloning and expression of squalene synthase and 2,3-oxidosqualene cyclase genes in persimmon (Diospyros kaki L.) fruits.

    PubMed

    Zhou, Chunhua; Zhao, Daqiu; Sheng, Yanle; Liang, Guohua; Tao, Jun

    2012-02-01

    Oleanolic acid (OA) and ursolic acid (UA) are the main triterpene acids in persimmon fruit, and squalene synthase and 2,3-oxidosqualene cyclases are important enzymes in pentacyclic triterpene biosynthesis. In order to study their relationship, DkSQS and DkOSC were cloned from persimmon fruits in the present study. The full-length cDNA of DkSQS was 1647 bp, containing an open reading frame (ORF) of 1245 bp that encoded a peptide of 415 amino acids (AA). The 3'-end of DkOSC cDNA fragment contained 522 bp, including a partial ORF of 298 bp, a full poly A tail that encoded 98 AA. Two cultivars of persimmon, i.e. cv. Nishimurawase and cv. Niuxinshi, were used to study the content of OA and UA and the related gene expression. Results showed that OA and UA contents changed in both cultivars during fruit development, the difference in cv. Nishimurawase was greater than that in cv. Niuxinshi. The expression of DkSQS and DkOSC had no obvious correlation with the biosynthesis of OA and UA in the flesh. There may be two main reasons. Firstly, different enzymes involved in the biosynthesis of triterpenes and mutual adjustment were existed in different gene expressions. Secondly, it was not clear that the DkOSC cloned in this research belonged to which subfamily. Therefore, the real relationship between triterpenes and DkSQS and DkOSC in persimmon fruits is still to be revealed.

  8. Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Krämer, Reinhard; Stephanopoulos, Gregory; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum. PMID:14711665

  9. Control of the ATP synthase beta subunit expression by RNA-binding proteins TIA-1, TIAR, and HuR.

    PubMed

    Izquierdo, José M

    2006-09-22

    The beta-subunit of the mitochondrial H+-ATP synthase (beta-F1-ATPase) catalyzes the rate-limiting step of ATP formation in eukaryotic cells. Here, we examined the post-transcriptional regulation of human beta-F1-ATPase mediated by the 3'-untranslated region of the mRNA (beta-3'-UTR). Biochemical analysis revealed that the adenosine/uridine (AU)-rich element-binding proteins TIA-1 (T-cell intracellular antigen-1), TIAR (TIA-1-related protein), and HuR (Hu antigen R) interact with the beta-F1-ATPase mRNA through an AU-rich sequence located to the 3'-UTR. Mouse embryonic fibroblasts (MEFs) knocked-out for TIA-1 or RNA interference (RNAi)-mediated knockdown of endogenous TIA-1, TIAR, or HuR in HeLa cells resulted in a decrease in beta-F1-ATPase protein expression. The expression of GFP from a chimeric reporter containing human beta-3'-UTR was also abolished in HeLa cells depleted of TIA-1, TIAR, or HuR. MEFs knocked-in for TIA-1 or the overexpression of RNAi-resistant TIA-1, TIAR, or HuR proteins in the RNAi-treated HeLa cells significantly restored the levels of the expression of both endogenous mouse beta-F1-ATPase protein or recombinant GFP.

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

    PubMed Central

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S; Parkos, Charles A.; Nusrat, Asma

    2010-01-01

    The apical junctional complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin. PMID:20617560

  11. Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit.

    PubMed

    Rodríguez-Concepción, Manuel; Querol, Jordi; Lois, Luisa María; Imperial, Santiago; Boronat, Albert

    2003-07-01

    Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato ( Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe ( r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

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

    SciTech Connect

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    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.

  13. Nitric Oxide Synthase Expression in Macrophages of Histoplasma capsulatum-Infected Mice Is Associated with Splenocyte Apoptosis and Unresponsiveness

    PubMed Central

    Wu-Hsieh, Betty A.; Chen, Wen; Lee, Hsin-Ju

    1998-01-01

    Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of NG-monomethyl-l-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice. PMID:9784566

  14. ACTIVATION OF VASCULAR ENDOTHELIAL NITRIC OXIDE SYNTHASE AND HEME OXYGENASE-1 EXPRESSION BY ELECTROPHILIC NITRO-FATTY ACIDS

    PubMed Central

    Khoo, Nicholas K.H.; Rudolph, Volker; Cole, Marsha P.; Golin-Bisello, Franca; Schopfer, Francisco J.; Woodcock, Steven R.; Batthyany, Carlos; Freeman, Bruce A.

    2010-01-01

    Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated byproducts of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yield electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO2) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO2 via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO2-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO2-FAs stimulated the phosphorylation of eNOS at Ser1179. These post-translational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO2-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders. PMID:19857569

  15. Enhanced phytoremediation of toxic metals by inoculating endophytic Enterobacter sp. CBSB1 expressing bifunctional glutathione synthase.

    PubMed

    Qiu, Zhiqi; Tan, Hongming; Zhou, Shining; Cao, Lixiang

    2014-02-28

    To engineer plant-bacteria symbionts for remediating complex sites contaminated with multiple metals, the bifunctional glutathione (GSH) synthase gene gcsgs was introduced into endophytic Enterobacter sp. CBSB1 to improve phytoremediation efficiency of host plant Brassica juncea. The GSH contents of shoots inoculated with CBSB1 is 0.4μMg(-1) fresh weight. However, the GSH concentration of shoots with engineered CBSB1-GCSGS increased to 0.7μMg(-1) fresh weight. The shoot length, fresh weight and dry weight of seedlings inoculated with CBSB1-GCSGS increased 67%, 123%, and 160%, compared with seedlings without inoculation, respectively. The Cd and Pb concentration in shoots with CBSB1-GCSGS increased 48% and 59% compared with seedlings without inoculation, respectively. The inoculation of CBSB1 and CBSB1-GCSGS could increase the Cd and Pb extraction amounts of seedlings significantly compared with those without inoculation (P<0.05), the seedlings inoculated with CBSB1-GCSGS showed the highest Cd and Pb extraction amounts. It was concluded that the gcsgs gene introduced into Enterobacter sp. CBSB1 upgraded the phytoremediation efficacy of B. juncea. So the engineered Enterobacter sp. CBSB1-GCSGS showed potentials in remediation sites contaminated with complex contaminants by inoculating into remediating plants.

  16. Nitric oxide synthase is expressed in human macrophages during foreign body inflammation.

    PubMed Central

    Moilanen, E.; Moilanen, T.; Knowles, R.; Charles, I.; Kadoya, Y.; al-Saffar, N.; Revell, P. A.; Moncada, S.

    1997-01-01

    Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes. Images Figure 1 Figure 2 Figure 3 PMID:9060826

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

    PubMed

    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.

  18. Endodontic-orthodontic relationships: expression of no synthase in human dental pulp during orthodontic tooth movement.

    PubMed

    D ' Attilio, M; De Angelis, F; Vadini, M; Rodolfino, D; Trubiani, O; Di Nardo Di Maio, F; D' Arcangelo, C

    2012-01-01

    Inflamed human pulp tissue presents an increase in the level of nitric oxide synthase (NOS). The aim of this study is to verify the presence of NOS in human pulp of teeth that are subject to orthodontic force. 20 healthy subjects, wearers of fixed braces on the upper arch, were selected. An open coil-spring in NiTi was applied on the upper premolar test tooth (TT); the controlateral control tooth (CCT) was subjected to orthodontic treatment but not to the further force of the open coil-spring; the antagonist control tooth (ACT) did not undergo any orthodontic treatment. Pulps were taken from test, contralateral control and antagonist control teeth immediately after the extractions which were done at 15 and 30 days from the start of application of the orthodontic force. The pulp tissue was analyzed through immunohistochemical and molecular biology examinations. The results showed tooth pulps subject to orthodontic treatment were very inflamed in the first 15 days with high levels of iNOS and low levels of eNOS; after 30 days a decrease of the inflammation and an increase of the pulp vascularization were observed together with a reduction of iNOS and an increase of eNOS respectively.

  19. Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

    PubMed

    Yao, Lin; Tan, Chong; Song, Jinzhu; Yang, Qian; Yu, Lijie; Li, Xinling

    2016-01-01

    Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

  20. Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis--phylogeny, site-directed mutagenesis, and expression in nonanther tissues.

    PubMed

    Jepson, Christina; Karppinen, Katja; Daku, Rhys M; Sterenberg, Brian T; Suh, Dae-Yeon

    2014-09-01

    Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C6) to very long-chain (C24) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C16-C18, and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

  1. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

    PubMed Central

    Mahmoud, Soheil S.; Croteau, Rodney B.

    2001-01-01

    Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition. PMID:11427737

  2. Mechanisms of renal control of potassium homeostasis in complete aldosterone deficiency.

    PubMed

    Todkar, Abhijeet; Picard, Nicolas; Loffing-Cueni, Dominique; Sorensen, Mads V; Mihailova, Marija; Nesterov, Viatcheslav; Makhanova, Natalia; Korbmacher, Christoph; Wagner, Carsten A; Loffing, Johannes

    2015-02-01

    Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted.

  3. Mechanisms of Renal Control of Potassium Homeostasis in Complete Aldosterone Deficiency

    PubMed Central

    Todkar, Abhijeet; Picard, Nicolas; Loffing-Cueni, Dominique; Sorensen, Mads V.; Mihailova, Marija; Nesterov, Viatcheslav; Makhanova, Natalia; Korbmacher, Christoph; Wagner, Carsten A.

    2015-01-01

    Aldosterone-independent mechanisms may contribute to K+ homeostasis. We studied aldosterone synthase knockout (AS−/−) mice to define renal control mechanisms of K+ homeostasis in complete aldosterone deficiency. AS−/− mice were normokalemic and tolerated a physiologic dietary K+ load (2% K+, 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K+ intake (5% K+), AS−/− mice decompensated and became hyperkalemic. High-K+ diets induced upregulation of the renal outer medullary K+ channel in AS−/− mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K+ excretion was detected only with a 2% K+ diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS−/− mice than in AS+/+ mice and was downregulated in mice of both genotypes in response to increased K+ intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS−/− mice. In contrast with the kidney, the distal colon of AS−/− mice did not respond to dietary K+ loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K+ load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K+ channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K+ secretion and increased intratubular availability of Na+ that can be reabsorbed in exchange for K+ secreted. PMID:25071088

  4. Expression and regulation of endothelial nitric oxide synthase by vascular endothelial growth factor in ECV 304 cells.

    PubMed Central

    Park, Jong Seon; Hong, Gu Ru; Baek, Suk Whan; Shin, Dong Gu; Kim, Young Jo; Shim, Bong Sup

    2002-01-01

    Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with VEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to VEGF(165), eNOS activity and cell growth were increased by approximately two-fold in the VEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, VEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway. PMID:11961297

  5. H2S regulates endothelial nitric oxide synthase protein stability by promoting microRNA-455-3p expression

    PubMed Central

    Li, Xing-Hui; Xue, Wen-Long; Wang, Ming-Jie; Zhou, Yu; Zhang, Cai-Cai; Sun, Chen; Zhu, Lei; Liang, Kun; Chen, Ying; Tao, Bei-Bei; Tan, Bo; Yu, Bo; Zhu, Yi-Chun

    2017-01-01

    The aims of the present study are to determine whether hydrogen sulfide (H2S) is involved in the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, and to identify the role of microRNA-455-3p (miR-455-3p) during those processes. In cultured human umbilical vein endothelial cells (HUVECs), the expression of miR-455-3p, eNOS protein and the NO production was detected after administration with 50 μM NaHS. The results indicated that H2S could augment the expression of miR-455-3p and eNOS protein, leading to the increase of NO level. We also found that overexpression of miR-455-3p in HUVECs increased the protein levels of eNOS whereas inhibition of miR-455-3p decreased it. Moreover, H2S and miR-455-3p could no longer increase the protein level of eNOS in the presence of proteasome inhibitor, MG-132. In vivo, miR-455-3p and eNOS expression were considerably increased in C57BL/6 mouse aorta, muscle and heart after administration with 50 μmol/kg/day NaHS for 7 days. We also identified that H2S levels and miR-455-3p expression increased in human atherosclerosis plaque while H2S levels decreased in plasma of atherosclerosis patients. Our data suggest that the stability of eNOS protein and the NO production could be regulated by H2S through miR-455-3p. PMID:28322298

  6. The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase.

    PubMed

    Baldini, Steffi F; Wavelet, Cindy; Hainault, Isabelle; Guinez, Céline; Lefebvre, Tony

    2016-08-14

    Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases.

  7. Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses.

    PubMed

    Gondim, Fernando J; Modolo, Luzia V; Campos, Gerson E R; Salgado, I

    2005-01-01

    Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.

  8. Early Growth Response1and Fatty Acid Synthase Expression is Altered in Tumor Adjacent Prostate Tissue and Indicates Field Cancerization

    PubMed Central

    Jones, Anna C.; Trujillo, Kristina A.; Phillips, Genevieve K.; Fleet, Trisha M.; Murton, Jaclyn K.; Severns, Virginia; Shah, Satyan K.; Davis, Michael S.; Smith, Anthony Y.; Griffith, Jeffrey K.; Fischer, Edgar G.; Bisoffi, Marco

    2011-01-01

    BACKGROUND Field cancerization denotes the occurrence of molecular alterations in histologically normal tissues adjacent to tumors. In prostate cancer, identification of field cancerization has several potential clinical applications. However, prostate field cancerization remains ill defined. Our previous work has shown up-regulated mRNA of the transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS) in tissues adjacent to prostate cancer. METHODS Immunofluorescence data were analyzed quantitatively by spectral imaging and linear unmixing to determine the protein expression levels of EGR-1 and FAS in human cancerous, histologically normal adjacent, and disease-free prostate tissues. RESULTS EGR-1 expression was elevated in both structurally intact tumor adjacent (1.6× on average) and in tumor (3.0× on average) tissues compared to disease-free tissues. In addition, the ratio of cytoplasmic versus nuclear EGR-1 expression was elevated in both tumor adjacent and tumor tissues. Similarly, FAS expression was elevated in both tumor adjacent (2.7× on average) and in tumor (2.5× on average) compared to disease-free tissues. CONCLUSIONS EGR-1 and FAS expression is similarly deregulated in tumor and structurally intact adjacent prostate tissues and defines field cancerization. In cases with high suspicion of prostate cancer but negative biopsy, identification of field cancerization could help clinicians target areas for repeat biopsy. Field cancerization at surgical margins on prostatectomy specimen should also be looked at as a predictor of cancer recurrence. EGR-1 and FAS could also serve as molecular targets for chemoprevention. PMID:22127986

  9. Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy

    PubMed Central

    Fath Bayati, Leyla; Ghaffari Novin, Marefat; Fadaei Fathabadi, Fatemeh; Piryaei, Abbas; Heidari, Mohammad Hasan; Bandehpour, Mozhgan; Norouzian, Mohsen; Alizadeh Parhizgar, Mahdi; Shakooriyan Fard, Mahmood

    2014-01-01

    Background: Tubal ectopic pregnancy (tEP) is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in the regulation of many reproductive events that occur in the fallopian tube (FT). Objective: The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. Materials and Methods: In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women (n=10). Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Results: Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase (p=0.00). Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups (p=0.78). Conclusion: This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP. This article extracted from M.Sc. thesis. (Leyla Fath Bayati) PMID:24799858

  10. Chrysanthemum expressing a linalool synthase gene 'smells good', but 'tastes bad' to western flower thrips.

    PubMed

    Yang, Ting; Stoopen, Geert; Thoen, Manus; Wiegers, Gerrie; Jongsma, Maarten A

    2013-09-01

    Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound.

  11. Primary aldosteronism and impaired natriuresis in mice underexpressing TGFβ1

    PubMed Central

    Kakoki, Masao; Pochynyuk, Oleh M.; Hathaway, Catherine M.; Tomita, Hirofumi; Hagaman, John R.; Kim, Hyung-Suk; Zaika, Oleg L.; Mamenko, Mykola; Kayashima, Yukako; Matsuki, Kota; Hiller, Sylvia; Li, Feng; Xu, Longquan; Grant, Ruriko; Bertorello, Alejandro M.; Smithies, Oliver

    2013-01-01

    To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor β1 (TGFβ1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na+, K+-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function. PMID:23503843

  12. Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice

    PubMed Central

    Sica, Monica; Martini, Mariangela; Viglietti-Panzica, Carla; Panzica, GianCarlo

    2009-01-01

    Background Nitric oxide plays an important role in the regulation of male and female sexual behavior in rodents, and the expression of the nitric oxide synthase (NOS) is influenced by testosterone in the male rat, and by estrogens in the female. We have here quantitatively investigated the distribution of nNOS immunoreactive (ir) neurons in the limbic hypothalamic region of intact female mice sacrificed during different phases of estrous cycle. Results Changes were observed in the medial preoptic area (MPA) (significantly higher number in estrus) and in the arcuate nucleus (Arc) (significantly higher number in proestrus). In the ventrolateral part of the ventromedial nucleus (VMHvl) and in the bed nucleus of the stria terminalis (BST) no significant changes have been observed. In addition, by comparing males and females, we observed a stable sex dimorphism (males have a higher number of nNOS-ir cells in comparison to almost all the different phases of the estrous cycle) in the VMHvl and in the BST (when considering only the less intensely stained elements). In the MPA and in the Arc sex differences were detected only comparing some phases of the cycle. Conclusion These data demonstrate that, in mice, the expression of nNOS in some hypothalamic regions involved in the control of reproduction and characterized by a large number of estrogen receptors is under the control of gonadal hormones and may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. PMID:19604366

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

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2015-01-01

    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). PMID:26057806

  14. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

    PubMed

    Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

    2015-08-07

    Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

  15. The Balance of Expression of Dihydroflavonol 4-reductase and Flavonol Synthase Regulates Flavonoid Biosynthesis and Red Foliage Coloration in Crabapples.

    PubMed

    Tian, Ji; Han, Zhen-yun; Zhang, Jie; Hu, YuJing; Song, Tingting; Yao, Yuncong

    2015-07-20

    Red leaf color is an attractive trait of Malus families, including crabapple (Malus spp.); however, little is known about the molecular mechanisms that regulate the coloration. Dihydroflavonols are intermediates in the production of both colored anthocyanins and colorless flavonols, and this current study focused on the gene expression balance involved in the relative accumulation of these compounds in crabapple leaves. Levels of anthocyanins and the transcript abundances of the anthocyanin biosynthetic gene, dihydroflavonol 4-reductase (McDFR) and the flavonol biosynthetic gene, flavonol synthase (McFLS), were assessed during the leaf development in two crabapple cultivars, 'Royalty' and 'Flame'. The concentrations of anthocyanins and flavonols correlated with leaf color and we propose that the expression of McDFR and McFLS influences their accumulation. Further studies showed that overexpression of McDFR, or silencing of McFLS, increased anthocyanin production, resulting in red-leaf and red fruit peel phenotypes. Conversely, elevated flavonol production and green phenotypes in crabapple leaves and apple peel were observed when McFLS was overexpressed or McDFR was silenced. These results suggest that the relative activities of McDFR and McFLS are important determinants of the red color of crabapple leaves, via the regulation of the metabolic fate of substrates that these enzymes have in common.

  16. The Balance of Expression of Dihydroflavonol 4-reductase and Flavonol Synthase Regulates Flavonoid Biosynthesis and Red Foliage Coloration in Crabapples

    PubMed Central

    Tian, Ji; Han, Zhen-yun; Zhang, Jie; Hu, YuJing; Song, Tingting; Yao, Yuncong

    2015-01-01

    Red leaf color is an attractive trait of Malus families, including crabapple (Malus spp.); however, little is known about the molecular mechanisms that regulate the coloration. Dihydroflavonols are intermediates in the production of both colored anthocyanins and colorless flavonols, and this current study focused on the gene expression balance involved in the relative accumulation of these compounds in crabapple leaves. Levels of anthocyanins and the transcript abundances of the anthocyanin biosynthetic gene, dihydroflavonol 4-reductase (McDFR) and the flavonol biosynthetic gene, flavonol synthase (McFLS), were assessed during the leaf development in two crabapple cultivars, ‘Royalty’ and ‘Flame’. The concentrations of anthocyanins and flavonols correlated with leaf color and we propose that the expression of McDFR and McFLS influences their accumulation. Further studies showed that overexpression of McDFR, or silencing of McFLS, increased anthocyanin production, resulting in red-leaf and red fruit peel phenotypes. Conversely, elevated flavonol production and green phenotypes in crabapple leaves and apple peel were observed when McFLS was overexpressed or McDFR was silenced. These results suggest that the relative activities of McDFR and McFLS are important determinants of the red color of crabapple leaves, via the regulation of the metabolic fate of substrates that these enzymes have in common. PMID:26192267

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

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2015-06-01

    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(1)2(1)2(1) and P12(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(1)21. The crystals of latent cgAUS1 belonged to space group P12(1)1 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).

  18. Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappaB binding to DNA.

    PubMed Central

    Park, S K; Lin, H L; Murphy, S

    1997-01-01

    Treatment of astroglial cells with interleukin 1beta and interferon gamma transcriptionally activates the nitric oxide synthase (NOS)-2 gene. The duration of mRNA expression is brief because of transcript instability. In addition, NO donors reduce the expression of NOS-2 mRNA dramatically by reducing the rate of transcription. In this study we observed that the NO donor, spermine NONOate did not inhibit the activation and translocation of NF-kappaB, a key transcription factor in the induction of NOS-2, but inhibited formation of the NF-kappaB-DNA complex. This effect was reversed by methaemoglobin (acting as an NO trap) and by the reducing agent dithiothreitol. Formation of the interferon-regulatory factor-DNA complex was unaffected by NO. These results suggest that NO can modulate its own production by interfering with NF-kappaB interaction with the promoter region of the NOS gene, a negative feedback effect that may be important for limiting NO production in vivo. PMID:9065784

  19. Sciatic nerve transection increases gluthatione antioxidant system activity and neuronal nitric oxide synthase expression in the spinal cord.

    PubMed

    Guedes, Renata Padilha; Dal Bosco, Lidiane; Araújo, Alex Sander da Rosa; Belló-Klein, Adriane; Ribeiro, Maria Flávia Marques; Partata, Wania Aparecida

    2009-12-16

    Glutathione (GSH) is a major non-enzymatic antioxidant which is present in all tissues. Its protective actions occur through different pathways such its role as a substrate of antioxidant enzymes, such as glutathione peroxidase (GPx) and glutathione-S-transferase (GST). Nitric oxide (NO) is involved in many physiological processes in the central nervous system, including nociception. In spite of much evidence concerning oxidative and nitrosative stress and neuropathic pain, the exact role of these molecules in pain processing is still unknown. Sciatic nerve transection (SNT) was employed to induce neuropathic pain in rats. Glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities, glutathione (GSH) content, GSH/GSSG ratio, nitric oxide metabolites (NOx) and neuronal nitric oxide synthase (nNOS) protein expression in the lumbosacral spinal cord were determined. All of these analyses were performed in the SNT and sham groups 1, 3, 7 and 15 days after surgery. There was an increase in GPx activity and in GSH content 3 days after surgery in both sham and SNT groups, but the GSH/GSSG ratio increased only in the SNT group in this time point. nNOS expression was upregulated 7 days post SNT. NOx was detected 1 day after surgery in sham and SNT groups, but at 7 and 15 days, the increase occurred only in SNT animals. These results support the role of the gluthatione system in pain physiology and highlight the involvement of NO as an important molecule related to nociception.

  20. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants.

    PubMed

    Sánchez-Aguayo, Inmaculada; Rodríguez-Galán, José Manuel; García, Remedios; Torreblanca, José; Pardo, José Manuel

    2004-12-01

    S-Adenosyl-L-methionine synthase (SAM; ATP: L-methionine adenosyltransferase, EC 2.5.1.6) catalyzes the biosynthesis of S-adenosyl-L-methionine (AdoMet), a universal methyl-group donor. This enzyme is induced by salinity stress in tomato (Lycopersicon esculentum Mill.). To elucidate the role of SAM and AdoMet in the adaptation of plants to a saline environment, the expression pattern and histological distribution of SAM was investigated in control and salt-stressed tomato plants. Immunohistochemical analysis showed that SAM proteins were expressed in all cell types and plant organs, albeit with preferential accumulation in lignified tissues. Lignin deposition was estimated by histochemical tests and the extent of tissue lignification in response to salinity was quantified by image analysis. The average number of lignified cells in vascular bundles was significantly greater in plants under salt stress, with a maximal expansion of the lignified area found in the root vasculature. Accordingly, the greatest abundance of SAM gene transcripts and proteins occurred in roots. These results indicate that increased SAM activity correlated with a greater deposition of lignin in the vascular tissues of plants under salinity stress. A model is proposed in which an increased number of lignified tracheary elements in tomato roots under salt stress may enhance the cell-to-cell pathway for water transport, which would impart greater selectivity and reduced ion uptake, and compensate for diminished bulk flow of water and solutes along the apoplastic pathway.

  1. Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

    PubMed

    Feng, Xiuyan; Zhang, Yiqian; Shao, Ningjun; Wang, Yanhui; Zhuang, Zhizhi; Wu, Ping; Lee, Matthew J; Liu, Yingli; Wang, Xiaonan; Zhuang, Jieqiu; Delpire, Eric; Gu, Dingying; Cai, Hui

    2015-05-15

    Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

  2. Renal sodium transporter/channel expression and sodium excretion in P2Y2 receptor knockout mice fed a high-NaCl diet with/without aldosterone infusion.

    PubMed

    Zhang, Yue; Listhrop, Raelene; Ecelbarger, Carolyn M; Kishore, Bellamkonda K

    2011-03-01

    The P2Y(2) receptor (P2Y2-R) antagonizes sodium reabsorption in the kidney. Apart from its effect in distal nephron, hypothetically, P2Y(2)-R may modulate activity/abundances of sodium transporters/channel subunits along the nephron via antagonism of aldosterone or vasopressin or interaction with mediators such as nitric oxide (NO), and prostaglandin E(2) (PGE(2)) or oxidative stress (OS). To determine the extent of the regulatory role of P2Y(2)-R in renal sodium reabsorption, in study 1, we fed P2Y(2)-R knockout (KO; n = 5) and wild-type (WT; n = 5) mice a high (3.15%)-sodium diet (HSD) for 14 days. Western blotting revealed significantly higher protein abundances for cortical and medullary bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), medullary α-1-subunit of Na-K-ATPase, and medullary α-subunit of the epithelial sodium channel (ENaC) in KO vs. WT mice. Molecular analysis of urine showed increased excretion of nitrates plus nitrites (NOx), PGE(2), and 8-isoprostane in the KO, relative to WT mice, supporting a putative role for these molecules in determining alterations of proteins involved in sodium transport along the nephron. To determine whether genotype differences in response to aldosterone might have played a role in these differences due to HSD, in study 2 aldosterone levels were clamped (by osmotic minipump infusion). Clamping aldosterone (with HSD) led to significantly impaired natriuresis with elevated Na/H exchanger isoform 3 in the cortex, and NKCC2 in the medulla, and modest but significantly lower levels of NKCC2, and α- and β-ENaC in the cortex of KO vs. WT mice. This was associated with significantly reduced urinary NOx in the KO, although PGE(2) and 8-isoprostane remained significantly elevated vs. WT mice. Taken together, our results suggest that P2Y(2)-R is an important regulator of sodium transporters along the nephron. Pre- or postreceptor differences in the response to aldosterone, perhaps mediated via prostaglandins or changes in

  3. Induction of aminolevulinic acid synthase gene expression and enhancement of metabolite, protoporphyrin IX, excretion by organic germanium.

    PubMed

    Nakamura, Takashi; Saito, Miki; Shimada, Yasuhiro; Fukaya, Haruhiko; Shida, Yasuo; Tokuji, Yoshihiko

    2011-02-25

    Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132 is a water-soluble organic germanium compound. Oral intake of dietary Ge-132 changes fecal color and we attempted to identify the fecal red pigment, which increased by the intake of dietary Ge-132. Sprague Dawley rats were given diets containing Ge-132 from 0 to 0.5% concentration. Fecal red pigment was extracted and purified for optical and structural studies. We examined the fecal red pigment content by high performance liquid chromatography (HPLC), and hepatic gene expressions relating to heme synthesis by reverse transcription polymerase chain reaction (RT-PCR). The purified red pigment had particular optical characteristics on the ultraviolet (UV)-visible spectrum (Soret band absorbance at 400 nm) and fluorescence emission at 600 nm by 400 nm excitation, and was identified as protoporphyrin IX by LC-MS analysis. Protoporphyrin IX significantly (P<0.05) increased 2.4-fold in the feces by the intake of a 0.5% Ge-132 diet. Gene expression analysis of the liver explained the increase of protoporphyrin IX by dietary Ge-132 as it enhanced (P<0.05) aminolevulinic acid synthase 1 (Alas1), a rate-limiting enzyme of heme synthesis, expression 1.8-fold, but decreased ferrochelatase (Fech) expression 0.6-fold (P<0.05). The results show that the intake of dietary Ge-132 is related to heme metabolism. Because protoporphyrin IX is used to treat chronic hepatitis, Ge-132 may be a beneficial substance to increase protoporphyrin IX in the liver.

  4. Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand.

    PubMed

    Sari, M A; Booker, S; Jaouen, M; Vadon, S; Boucher, J I; Pompon, D; Mansuy, D

    1996-06-04

    Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

  5. Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability

    PubMed Central

    Suschek, Christoph Viktor; Bonmann, Eckhard; Kleinert, Hartmut; Wenzel, Michael; Mahotka, Csaba; Kolb, Hubert; Förstermann, Ulrich; Gerharz, Claus-Dieter; Kolb-Bachofen, Victoria

    2000-01-01

    The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures.AmB used at concentrations of 0.6 to 1.25 μg ml−1 led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5–5.0 μg ml−1) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells.Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity. PMID:11015297

  6. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  7. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    PubMed

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (p<0.01) higher serum NO production in ghrelin treated HF rats compared with HF rats. Ghrelin significantly reduced citrulline concentration (p<0.05) and arginase activity (p<0.01) in HF rats. In ghrelin treated HF rats, gene and protein expression of iNOS and NFκB-p65 levels were significantly (p<0.05) increased compared with HF rats. Increased phosphorylation of Akt (p<0.01) and decreased (p<0.05) ERK1/2 phosphorylation were detected in HF ghrelin treated rats compared with HF rats hearts.Results from this study indicate that exogenous ghrelin induces expression and activity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats.

  8. The Influence of Vitamin D Treatment on the Inducible Nitric Oxide Synthase (INOS) Expression in Primary Hippocampal Neurons

    PubMed Central

    DURSUN, Erdinç; GEZEN-AK, Duygu; YILMAZER, Selma

    2014-01-01

    Introduction Neurodegeneration is a process that is characterized by the loss of neuronal structure and function and eventually ends with neuronal death. An elevated level of inducible nitric oxide synthase (iNOS) is suggested to accompany this process by inducing oxidative and nitrosative damage. Vitamin D is reported to protect glial cells against neurotoxicity via suppressing iNOS synthesis. Though there was no data about whether iNOS is regulated by vitamin D in hippocampal neurons. In this study our aim was to determine any alteration in iNOS expression of hippocampal neurons in response to vitamin D treatment. Method Twenty four and 48 hours of vitamin D treatments were performed on primary hippocampal neuron cultures that were prepared from Sprague dawley rat embryos (E18). The alterations in the iNOS mRNA expression were determined with quantative real time polymerase chain reaction (qRT-PCR). The cytotoxicity levels of each group were investigated by the measurement of lactate dehydrogenase (LDH) that is released to culture medium. Results No difference was observed between groups in 24 hours of treatment regarding the iNOS expression. Though the iNOS mRNA level of vitamin D treated group was significantly lower than that of control group on the 48th hours of treatment (p<.001). Vitamin D treatment also attenuated the LDH release which is an indicator of cytotoxicity (p<.001). Conclusion Our results indicated that vitamin D has the potential to prevent oxidative damage by suppressing iNOS expression.

  9. GNC and CGA1 modulate chlorophyll biosynthesis and glutamate synthase (GLU1/Fd-GOGAT) expression in Arabidopsis.

    PubMed

    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.

  10. Preoperative Chemoradiation for Rectal Cancer Using Capecitabine and Celecoxib Correlated With Posttreatment Assessment of Thymidylate Synthase and Thymidine Phosphorylase Expression

    SciTech Connect

    Unger, Keith R.; Romney, Davis A.; Koc, Mehmet; Moskaluk, Christopher A.; Friel, Charles M.; Foley, E.F.; Rich, Tyvin A.

    2011-08-01

    Purpose: Thymidylate synthase (TS) and thymidine phosphorylase (TP) expression have been shown to be predictors of response to therapy. The toxicity, efficacy, surgical morbidity, and immunohistochemical TS and TP expression were assessed in surgical resection specimens after preoperative chemoradiation. Methods and Materials: Twenty patients with clinical stage I to III rectal adenocarcinoma received preoperative chemoradiation and underwent surgical resection 6 weeks later. Results: Posttreatment tumor stages were T1 to T2 and N0 in 30% of patients; T3 to T4 and N0 in 30% of patients; and T1 to T3 and N1 to N2 in 15% of patients. Pathologic complete response (pCR) was evident in 25% and tumor regression occurred in a total of 80% of patients. Anal sphincter-sparing surgery was performed in 80% of cases. Acute and perioperative complications were minimal, with no grade 3/4 toxicity or treatment breaks. Pelvic control was obtained in 90% of patients. With a median follow-up of 65.5 months (range, 8-80 months), the 6-year actuarial survival rate was 75%. Local failure was significantly associated with nonresponse to therapy and with high TS and low TP expression (p = 0.008 and p = 0.04, respectively). Conclusions: The combination of capecitabine, celecoxib, and x-radiation therapy yields excellent response: a 25% pathologic pCR, no acute grade 3/4 toxicity, and minimal surgical morbidity. Nonresponders expressed significantly increased TS levels and decreased TP levels in posttreatment resection specimens compared to responders.

  11. Inducible nitric oxide synthase response and associated cytokine gene expression in the spleen of mice infected with Clonorchis sinensis.

    PubMed

    Shen, Ji-Qing; Yang, Qing-Li; Xue, Yan; Cheng, Xiao-Bing; Jiang, Zhi-Hua; Yang, Yi-Chao; Chen, Ying-Dan; Zhou, Xiao-Nong

    2015-05-01

    Clonorchis sinensis is a food-borne parasite that induces a permanent increase of nitrosation in the body upon infection. The spleen is an important secondary lymphoid organ for the regulation of immune responses locally and in the whole body. However, the functions and mechanisms of the spleen in nitric oxide (NO) responses after C. sinensis infection remain unknown. In this study, BALB/c mice were infected with 20, 40, and 80 C. sinensis metacercariae to simulate mild, moderate, and severe infections, respectively. We examined the expression of inducible nitric oxide synthase (iNOS) in the spleen and the relevant cytokine transcription in splenocytes from the mice infected with different amounts of metacercariae. The iNOS of the mice infected with 80 metacercariae was expressed in the spleen as early as 10 days post-infection (dpi) and gradually increased until 90 dpi. The iNOS expression in the mice infected with 40 metacercariae was detected only at 45 and 90 dpi, but not in the mice infected with 20 metacercariae. The level of interferon (IFN)-γ messenger RNA (mRNA) transcription in splenocytes significantly increased at 10 and 20 dpi (P < 0.05) in response to mild/moderate infection but gradually decreased to normal levels after 45 dpi. The level of IL-12p35 mRNA transcription did not change at 10 and 20 dpi but significantly decreased after 45 dpi under moderate/severe infection (P < 0.05/0.01/0.001). The level of IL-18 mRNA transcription significantly increased at 10 dpi (P < 0.05/0.01) but significantly decreased after 20 dpi (P < 0.05/0.01/0.001). These results suggest that spleen is an important organ for iNOS/NO responses, which correspond to the severity of C. sinensis infection, but cannot be attributed to the expression of the Th1 cytokines.

  12. Nitric-oxide synthase is a mechanical signal transducer that modulates talin and vinculin expression

    NASA Technical Reports Server (NTRS)

    Tidball, J. G.; Spencer, M. J.; Wehling, M.; Lavergne, E.

    1999-01-01

    Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

  13. An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

    PubMed Central

    Wang, Yang; Newton, Derek C.; Miller, Tricia L.; Teichert, Anouk-Martine; Phillips, M. James; Davidoff, Michail S.; Marsden, Philip A.

    2002-01-01

    Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of β-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. β-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P450scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, β-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of β-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, β-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of

  14. [Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa].

    PubMed

    Wang, Bingjie; Li, Houhua; Wang, Yajie; Gaol, Yan; Fu, Wany; Weil, Xincui

    2015-12-01

    Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the niain active medicinal compound for cancer prevention and treatment. A full-length FNSII gene, namely SmFNSII (GenBank Accession No. KF170286), was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that SmFNSII is 1 710 bp in full length, containing a 34 bp 5'-untranslated region (5'-UTR), a 125 bp 3'-UTR, and a 1 551 bp open reading frame (ORF) encoding 516 amino acid residues. Amino acid sequence analysis indicated that SmFNSII belonged to subfamily CYP93B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of SmFNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that SmFNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. pET-SmFNSII, a prokaryotic expression recombinant plasmid, was constructed and transferred into Escherichia coli, and the expressed protein band was the same size with predicted protein. Saussurea medusa cultivars with high anti-inflammatory, anti-cancer activities and health care function would be cultivated through filtering cell lines and plants with high expression level of FNSII gene and luteolin accumulation.

  15. Sleep active cortical neurons expressing neuronal nitric oxide synthase are active after both acute sleep deprivation and chronic sleep restriction.

    PubMed

    Zielinski, M R; Kim, Y; Karpova, S A; Winston, S; McCarley, R W; Strecker, R E; Gerashchenko, D

    2013-09-05

    Non-rapid eye movement (NREM) sleep electroencephalographic (EEG) delta power (~0.5-4 Hz), also known as slow wave activity (SWA), is typically enhanced after acute sleep deprivation (SD) but not after chronic sleep restriction (CSR). Recently, sleep-active cortical neurons expressing neuronal nitric oxide synthase (nNOS) were identified and associated with enhanced SWA after short acute bouts of SD (i.e., 6h). However, the relationship between cortical nNOS neuronal activity and SWA during CSR is unknown. We compared the activity of cortical neurons expressing nNOS (via c-Fos and nNOS immuno-reactivity, respectively) and sleep in rats in three conditions: (1) after 18-h of acute SD; (2) after five consecutive days of sleep restriction (SR) (18-h SD per day with 6h ad libitum sleep opportunity per day); (3) and time-of-day matched ad libitum sleep controls. Cortical nNOS neuronal activity was enhanced during sleep after both 18-h SD and 5 days of SR treatments compared to control treatments. SWA and NREM sleep delta energy (the product of NREM sleep duration and SWA) were positively correlated with enhanced cortical nNOS neuronal activity after 18-h SD but not 5days of SR. That neurons expressing nNOS were active after longer amounts of acute SD (18h vs. 6h reported in the literature) and were correlated with SWA further suggest that these cells might regulate SWA. However, since these neurons were active after CSR when SWA was not enhanced, these findings suggest that mechanisms downstream of their activation are altered during CSR.

  16. Disequilibrium of Flavonol Synthase and Dihydroflavonol-4-Reductase Expression Associated Tightly to White vs. Red Color Flower Formation in Plants

    PubMed Central

    Luo, Ping; Ning, Guogui; Wang, Zhen; Shen, Yuxiao; Jin, Huanan; Li, Penghui; Huang, Shasha; Zhao, Jian; Bao, Manzhu

    2016-01-01

    Flower color is the main character throughout the plant kingdom. Though substantial information exists regarding the structural and regulatory genes involved in anthocyanin and flavonol biosynthesis, little is known that what make a diverse white vs. red color flower in natural species. Here, the contents of pigments in seven species from varied phylogenetic location in plants with red and white flowers were determined. Flavonols could be detected in red and white flowers, but anthocyanins were almost undetectable in the white cultivar. Comparisons of expression patterns of gene related to the flavonoid biosynthesis indicated that disequilibrium expression of flavonol synthase (FLS) and dihydroflavonol-4-reductase (DFR) genes determined the accumulation of flavonols and anothcyanins in both red and white flowers of seven species. To further investigate the role of such common regulatory patterns in determining flower color, FLS genes were isolated from Rosa rugosa (RrFLS1), Prunus persica (PpFLS), and Petunia hybrida (PhFLS), and DFR genes were isolated from Rosa rugosa (RrDFR1) and Petunia hybrida (PhDFR). Heterologous expression of the FLS genes within tobacco host plants demonstrated conservation of function, with the transgenes promoting flavonol biosynthesis and inhibiting anthocyanin accumulation, so resulting in white flowers. Conversely, overexpression of DFR genes in tobacco displayed down-regulation of the endogenous NtFLS gene, and the promotion of anthocyanin synthesis. On this basis, we propose a model in which FLS and DFR gene-products compete for common substrates in order to direct the biosynthesis of flavonols and anthocyanins, respectively, thereby determining white vs. red coloration of flowers. PMID:26793227

  17. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    PubMed

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

  18. Testosterone induction of prostaglandin-endoperoxide synthase 2 expression and prostaglandin F(2alpha) production in hamster Leydig cells.

    PubMed

    Matzkin, María E; Gonzalez-Calvar, Silvia I; Mayerhofer, Artur; Calandra, Ricardo S; Frungieri, Mónica B

    2009-07-01

    We have previously observed expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the key enzyme in the biosynthesis of prostaglandins (PGs), in reproductively active Syrian hamster Leydig cells, and reported an inhibitory role of PGF(2alpha) on hamster testicular steroidogenesis. In this study, we further investigated PTGS2 expression in hamster Leydig cells during sexual development and photoperiodic gonadal regression. Since PTGS2 is mostly expressed in pubertal and reproductively active adult hamsters with high circulating levels of LH and androgens, we studied the role of these hormones in the regulation/maintenance of testicular PTGS2/PGF(2alpha). In active hamster Leydig cells, LH/hCG and testosterone induced PTGS2 and PGF(2alpha) production, and their actions were abolished by the antiandrogen bicalutamide (Bi). These results indicate that LH does not exert a direct effect on PG synthesis. Testosterone also stimulated phosphorylation of the mitogen-activated protein kinase isoforms 3/1 (MAPK3/1) within minutes and hours, but the testosterone metabolite dihydrotestosterone had no effect on PTGS2 and MAPK3/1. Because Bi and U0126, an inhibitor of the MAP kinase kinases 1 and 2 (MAP2K1/2), abolished testosterone actions on MAPK3/1 and PTGS2, our studies suggest that testosterone directly induces PTGS2/PGF(2alpha) in hamster Leydig cells via androgen receptors and a non-classical mechanism that involves MAPK3/1 activation. Since PGF(2alpha) inhibits testosterone production, it might imply the existence of a regulatory loop that is setting a brake on steroidogenesis. Thus, the androgen environment might be crucial for the regulation of testicular PG production at least during sexual development and photoperiodic variations in hamsters.

  19. RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

    PubMed

    Baumgardt, Kathrin; Charoenpanich, Pornsri; McIntosh, Matthew; Schikora, Adam; Stein, Elke; Thalmann, Sebastian; Kogel, Karl-Heinz; Klug, Gabriele; Becker, Anke; Evguenieva-Hackenberg, Elena

    2014-04-01

    Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-β-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.

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

    PubMed Central

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

    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-1; group A, 300 mg.kg-1; group B, and only normal saline; group C (control group). They were mated with untreated females and the reproductive and chemical parameters were assessed for each group, including semen quality, serum testosterone, sperm fertility, gonad and body weight, serum NO concentration (by the Griess method), and iNOS gene expression (using RT-PCR). Results: The testes weight, serum testosterone, as well as sperm count and fertility of the AVG treated groups were significantly reduced when compared to the control (P<0.001). Concentration of serum NO was significantly increased (37.1±4.63 µM) in the administrated group with higher AVG concentration, compared to the control group (P<0.001; 10.19±0.87 µM); however, iNOS mRNA expression was increased in the treated animals (P<0.001). Conclusion: iNOS may play a functional role in spermatogenesis via apoptosis, reducing sperm count, but further studies are needed to illustrate the mechanisms by which AVG exerts its negative effects on spermatogenesis and sperm quality. PMID:26730330

  1. Expression of Biphenyl Synthase Genes and Formation of Phytoalexin Compounds in Three Fire Blight-Infected Pyrus communis Cultivars

    PubMed Central

    Chizzali, Cornelia; Swiddan, Asya K.; Abdelaziz, Sahar; Gaid, Mariam; Richter, Klaus; Fischer, Thilo C.; Liu, Benye; Beerhues, Ludger

    2016-01-01

    Pear (Pyrus communis) is an economically important fruit crop. Drops in yield and even losses of whole plantations are caused by diseases, most importantly fire blight which is triggered by the bacterial pathogen Erwinia amylovora. In response to the infection, biphenyls and dibenzofurans are formed as phytoalexins, biosynthesis of which is initiated by biphenyl synthase (BIS). Two PcBIS transcripts were cloned from fire blight-infected leaves and the encoded enzymes were characterized regarding substrate specificities and kinetic parameters. Expression of PcBIS1 and PcBIS2 was studied in three pear cultivars after inoculation with E. amylovora. Both PcBIS1 and PcBIS2 were expressed in ‘Harrow Sweet’, while only PcBIS2 transcripts were detected in ‘Alexander Lucas’ and ‘Conference’. Expression of the PcBIS genes was observed in both leaves and the transition zone of the stem; however, biphenyls and dibenzofurans were only detected in stems. The maximum phytoalexin level (~110 μg/g dry weight) was observed in the transition zone of ‘Harrow Sweet’, whereas the concentrations were ten times lower in ‘Conference’ and not even detectable in ‘Alexander Lucas’. In ‘Harrow Sweet’, the accumulation of the maximum phytoalexin level correlated with the halt of migration of the transition zone, whereby the residual part of the shoot survived. In contrast, the transition zones of ‘Alexander Lucas’ and ‘Conference’ advanced down to the rootstock, resulting in necrosis of the entire shoots. PMID:27410389

  2. Tissue-specific expression and dietary regulation of chimeric mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice.

    PubMed

    Serra, D; Fillat, C; Matas, R; Bosch, F; Hegardt, F G

    1996-03-29

    We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.

  3. autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function.

    PubMed

    Vo, Phuong A; Lad, Bhavini; Tomlinson, James A P; Francis, Stephanie; Ahluwalia, Amrita

    2005-02-25

    Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.

  4. Altered sucrose synthase and invertase expression affects the local and systemic sugar metabolism of nematode-infected Arabidopsis thaliana plants.

    PubMed

    Cabello, Susana; Lorenz, Cindy; Crespo, Sara; Cabrera, Javier; Ludwig, Roland; Escobar, Carolina; Hofmann, Julia

    2014-01-01

    Sedentary endoparasitic nematodes of plants induce highly specific feeding cells in the root central cylinder. From these, the obligate parasites withdraw all required nutrients. The feeding cells were described as sink tissues in the plant's circulation system that are supplied with phloem-derived solutes such as sugars. Currently, there are several publications describing mechanisms of sugar import into the feeding cells. However, sugar processing has not been studied so far. Thus, in the present work, the roles of the sucrose-cleaving enzymes sucrose synthases (SUS) and invertases (INV) in the development of Heterodera schachtii were studied. Gene expression analyses indicate that both enzymes are regulated transcriptionally. Nematode development was enhanced on multiple INV and SUS mutants. Syncytia of these mutants were characterized by altered enzyme activity and changing sugar pool sizes. Further, the analyses revealed systemically affected sugar levels and enzyme activities in the shoots of the tested mutants, suggesting changes in the source-sink relationship. Finally, the development of the root-knot nematode Meloidogyne javanica was studied in different INV and SUS mutants and wild-type Arabidopsis plants. Similar effects on the development of both sedentary endoparasitic nematode species (root-knot and cyst nematode) were observed, suggesting a more general role of sucrose-degrading enzymes during plant-nematode interactions.

  5. Ceramides promote apoptosis for virus-infected lymphoma cells through induction of ceramide synthases and viral lytic gene expression

    PubMed Central

    Dai, Lu; Trillo-Tinoco, Jimena; Bai, Aiping; Chen, Yihan; Bielawski, Jacek; Del Valle, Luis; Smith, Charles D.; Ochoa, Augusto C.; Qin, Zhiqiang; Parsons, Chris

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for several human cancers including primary effusion lymphoma (PEL), a rapidly progressive malignancy arising preferentially in immunocompromised patients. With conventional chemotherapy, PEL continues to portend high mortality, dictating the development of novel therapeutic strategies. Sphingosine kinase 2 (SphK2) represents a key gatekeeper for sphingolipid metabolism, responsible for conversion of ceramides to sphingosine-1-phosphate (S1P). We have previously demonstrated that targeting SphK2 using a novel selective inhibitor, ABC294640, leads to intracellular accumulation of ceramides and induces apoptosis for KSHV-infected PEL cells, while suppressing tumor progression in vivo. In the current study, we sought to determine whether specific ceramide/dh-ceramide species and related ceramide synthases (CerS) impact viability for KSHV-infected PEL cells during targeting of SphK2. We found that several specific ceramide and dihydro(dh)-ceramide species and their associated CerS reduce PEL survival and tumor expansion in vitro and in vivo. Moreover, we found that dhC16-Cer induces PEL apoptosis in part through activation of KSHV lytic gene expression. These data further implicate bioactive sphingolipids in regulation of PEL survival, and provide justification for future studies evaluating clinically relevant ceramide analogs or mimetics for their potential as therapeutic agents for PEL. PMID:26327294

  6. [Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis].

    PubMed

    Liang, Liang; Guo, Qing-Mei; Zhang, Zheng; Xu, Yan-Hong; Han, Xiao-Min; Liu, Juan

    2014-12-01

    A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis

  7. Nitric oxide synthase expression in the central nervous system of Sepia officinalis: an in situ hybridization study.

    PubMed

    Di Cristo, Carlo; Fiore, Gabriella; Scheinker, Vladimir; Enikolopov, Grigori; d'Ischia, Marco; Palumbo, Anna; Di Cosmo, Anna

    2007-09-01

    We recently reported the molecular cloning of nitric oxide synthase (NOS) mRNA from Sepia officinalis (SoNOS) using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with a RACE procedure. Here, in situ hybridization study has been performed on serial sections of the cuttlefish central nervous system to reveal localized specific staining of cell bodies in several lobes of the brain. Staining was found in many lower motor centres, including cells of the inferior and superior buccal lobes (feeding centres); in some higher motor centres (anterior basal and peduncle lobes); in learning centres (vertical, subvertical and superior frontal lobes); and in the visual system [medulla and deep retina (optic lobe)]. Positive staining was also found in the olfactory lobe. NOS-expressing cells have been detected also in the interbasal lobe. Double labelling experiments, performed on consecutive sections, showed that neurons containing NOS immunoreactivity were also positive in in situ hybridization staining. All these data support the presence of NOS in several systems in the cuttlefish brain.

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

    PubMed

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

    2006-09-01

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

  9. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    SciTech Connect

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  10. 17beta-estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases.

    PubMed

    Noris, M; Todeschini, M; Zappella, S; Bonazzola, S; Zoja, C; Corna, D; Gaspari, F; Marchetti, G; Aiello, S; Remuzzi, G; Marchetti, F

    2000-10-01

    Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17beta-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor L-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17beta-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17beta-estradiol staining was comparable to controls. Thus 17beta-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.

  11. Characteristics of aldosterone binding in rat and human serum.

    PubMed

    Coirini, H; White, A; Marusic, E T; De Nicola, A F

    1982-01-01

    Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.

  12. Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

    PubMed Central

    Al-Hashmi, Sulaiman; Boels, Piet J. M.; Zadjali, Fahad; Sadeghi, Behnam; Sällström, Johan; Hultenby, Kjell; Hassan, Zuzana; Arner, Anders; Hassan, Moustapha

    2012-01-01

    Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late

  13. Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.).

    PubMed

    Qin, Xiaoqiong; Coku, Ardian; Inoue, Kentaro; Tian, Li

    2011-10-01

    Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

  14. Differential stress-response expression of two flavonol synthase genes and accumulation of flavonols in tartary buckwheat.

    PubMed

    Li, Xiaohua; Kim, Yeon Bok; Kim, Yeji; Zhao, Shicheng; Kim, Haeng Hoon; Chung, Eunsook; Lee, Jai-Heon; Park, Sang Un

    2013-12-15

    Flavonoids are ubiquitously present in plants and play important roles in these organisms as well as in the human diet. Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, acting at the diverging point into the flavonol subclass branch. We isolated and characterized a FLS isoform gene, FtFLS2, from tartary buckwheat (Fagopyrum tataricum). FtFLS2 shares 48% identity and 67% similarity with the previously reported FtFLS1, whereas both genes share 47-65% identity and 65-69% similarity with FLSs from other plant species. Using quantitative real-time PCR and high-performance liquid chromatography (HPLC), the expression of FtFLS1/2 and the production of 3 main flavonols (kaempferol, myricetin and quercetin) was detected in roots, leaves, stems, flowers and different stages of developing seeds. The relationship between the expression of the 2 FLS genes and the accumulation of the 3 basic flavonols was analyzed in 2 tartary buckwheat cultivars. FtFLS1 and FtFLS2 exhibited differential transcriptional levels between the tartary buckwheat cultivars 'Hokkai T10' and 'Hokkai T8'. Generally, higher transcript levels of FtFLS1 and FtFLS2 and a higher amount of flavonols were observed in the 'Hokkai T10' cultivar than 'Hokkai T8'. The content of flavonols showed tissue-specific accumulation between the 2 cultivars. The transcription of FtFLS1 was inhibited by the exogenous application of abscisic acid (ABA), salicylic acid (SA) and sodium chloride (NaCl), while FtFLS2 was not affected by ABA but up-regulated by SA and NaCl. These data indicate that the 2 FtFLS isoforms of buckwheat have different functions in the response of buckwheat to environmental stress.

  15. Expression and regulation of cytosolic prostaglandin E synthase in mouse uterus during the peri-implantation period.

    PubMed

    Ni, Hua; Sun, Tong; Ma, Xing-Hong; Yang, Zeng-Ming

    2003-03-01

    Prostaglandin E(2) (PGE(2)) is considered important for blastocyst spacing, implantation, and decidualization in rodent uteri. PGE synthase (PGES) catalyzes the isomerization of PGH(2) to PGE(2). Two isoforms of PGES exist: microsomal PGES (mPGES) and cytosolic PGES (cPGES); however, the expression and regulation of cPGES in the mammalian uterus during early pregnancy are still unknown. The aim of this study was to investigate the differential expression of cPGES in mouse uterus during early pregnancy and its regulation under different conditions using in situ hybridization and immunohistochemistry. A strong level of cPGES mRNA signal was exhibited in the stromal cells at the implantation site on Day 5 of pregnancy, whereas cPGES immunostaining was strongly detected in the luminal epithelium. The signals for both cPGES mRNA and immunostaining were strongly detected in the decidualized cells from Days 6-8 of pregnancy. A basal level of cPGES mRNA signal and immunostaining was exhibited in the uterus in delayed implantation. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, cPGES mRNA signal was strongly detected in the stroma underlying the luminal epithelium at the implantation site, and cPGES immunostaining was strongly observed in the luminal epithelium surrounding the implanting blastocyst. A strong cPGES mRNA signal and immunostaining were detected in decidualized cells under artificial decidualization, whereas only a basal level of cPGES mRNA signal and immunostaining were observed in the control horn. Our data suggest that cPGES may play an important role during implantation and decidualization.

  16. Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression.

    PubMed

    Lu, Shan; Xu, Ran; Jia, Jun-Wei; Pang, Jihai; Matsuda, Seiichi P T; Chen, Xiao-Ya

    2002-09-01

    Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.

  17. Potential for Quantifying Expression of the Geobacteraceae Citrate Synthase Gene To Assess the Activity of Geobacteraceae in the Subsurface and on Current-Harvesting Electrodes

    PubMed Central

    Holmes, Dawn E.; Nevin, Kelly P.; O'Neil, Regina A.; Ward, Joy E.; Adams, Lorrie A.; Woodard, Trevor L.; Vrionis, Helen A.; Lovley, Derek R.

    2005-01-01

    The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene. PMID:16269721

  18. Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes

    USGS Publications Warehouse

    Holmes, Dawn E.; Nevin, Kelly P.; O'Neil, Regina A.; Ward, Joy E.; Adams, Lorrie A.; Woodard, Trevor L.; Vrionis, Helen A.; Lovely, Derek R.

    2005-01-01

    The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene.

  19. Differential expression of (dihydro)ceramide synthases in mouse brain: oligodendrocyte-specific expression of CerS2/Lass2.

    PubMed

    Becker, Ivonne; Wang-Eckhardt, Lihua; Yaghootfam, Afshin; Gieselmann, Volkmar; Eckhardt, Matthias

    2008-02-01

    Synthesis of dihydroceramide is catalyzed by a family of (dihydro)ceramide synthases (CerS), first identified in yeast as longevity-assurance genes. Six members (CerS1-6; Lass1-6) of this gene family have been identified in mammals. We examined expression of CerS genes during postnatal development in mouse brain by means of Northern blot analysis, real-time RT-PCR, and in situ-hybridization. In situ-hybridization experiments showed that CerS1 was the predominant CerS in neurons throughout the brain. This observation is in line with the high levels of C18:0-ceramide in neurons and the substrate specificity of CerS1. A similar distribution, but lower expression levels, were found for CerS4 and CerS6. Only low or undetectable amounts of CerS1, CerS4 and CerS6 were, however, present in white matter. In contrast, CerS5 mRNA was detected in most cells within gray and white matter of all brain regions, suggesting ubiquitous expression of this palmitoyl-CoA specific CerS. Expression of CerS2 was transiently increased during the period of active myelination. Furthermore, expression of CerS2 was specifically localized to white matter tracts of the brain. Furthermore, CerS2 was the predominant CerS in Schwann cells of sciatic nerves. These data suggest that CerS2 is important for the synthesis of dihydroceramide used for synthesis of myelin sphingolipids.

  20. Aldosterone induces fibrosis, oxidative stress and DNA damage in livers of male rats independent of blood pressure changes.

    PubMed

    Queisser, Nina; Happ, Kathrin; Link, Samuel; Jahn, Daniel; Zimnol, Anna; Geier, Andreas; Schupp, Nicole

    2014-11-01

    Mineralocorticoid receptor blockers show antifibrotic potential in hepatic fibrosis. The mechanism of this protective effect is not known yet, although reactive oxygen species seem to play an important role. Here, we investigated the effects of elevated levels of aldosterone (Ald), the primary ligand of the mineralocorticoid receptor, on livers of rats in a hyperaldosteronism model: aldosterone-induced hypertension. Male Sprague-Dawley rats were treated for 4 weeks with aldosterone. To distinguish if damage caused in the liver depended on increased blood pressure or on increased Ald levels, the mineralocorticoid receptor antagonist spironolactone was given in a subtherapeutic dose, not normalizing blood pressure. To investigate the impact of oxidative stress, the antioxidant tempol was administered. Aldosterone induced fibrosis, detected histopathologically, and by expression analysis of the fibrosis marker, α-smooth muscle actin. Further, the mRNA amount of the profibrotic cytokine TGF-β was increased significantly. Fibrosis could be reduced by scavenging reactive oxygen species, and also by blocking the mineralocorticoid receptor. Furthermore, aldosterone treatment caused oxidative stress and DNA double strand breaks in livers, as well as the elevation of DNA repair activity. An increase of the transcription factor Nrf2, the main regulator of the antioxidative response could be observed, and of its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. All these effects of aldosterone were prevented by spironolactone and tempol. Already after 4 weeks of treatment, aldosteroneinfusion induced fibrosis in the liver. This effect was independent of elevated blood pressure. DNA damage caused by aldosterone might contribute to fibrosis progression when aldosterone is chronically increased.

  1. Engineering Fungal Nonreducing Polyketide Synthase by Heterologous Expression and Domain Swapping

    SciTech Connect

    Yeh, Hsu-Hua; Chang, Shu-Lin; Chiang, Yi-Ming; Bruno, Kenneth S.; Oakley, Berl R.; Wu, Tung-Kung; Wang, Clay C. C.

    2013-02-15

    Heterologous expression of the A. niger NR-PKS gene, e_gw1_19.204 and the adjacent stand-alone R domain gene, est_GWPlus_C_190476 in A. nidulans demonstrated that they belong to a single gene named dtbA. The DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde 1 and 2-ethyl-4,6-dihydroxy-3,5-dimethylbenzaldehyde 2. Generation of DtbA+R-TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes.

  2. Analysis of the mitochondrial ATP synthase beta-subunit gene in Drosophilidae: structure, transcriptional regulatory features and developmental pattern of expression in Drosophila melanogaster.

    PubMed Central

    Peña, P; Ugalde, C; Calleja, M; Garesse, R

    1995-01-01

    We have cloned and determined the structure of the gene encoding the H(+)-ATP synthase beta subunit in two distantly related Drosophila species, D. melanogaster and D. virilis. The gene contains three exons that are extremely well conserved at the amino acid level, not only in the region encoding the mature protein but also in that encoding the leader peptide. Primer extension analysis indicates that the 5' untranslated region is extremely short, and reveals the presence of multiple initiation sites of transcription in both Drosophila species. The promoters of D. melanogaster and D. virilis H(+)-ATP synthase beta-subunit genes contain a conserved region surrounding the initiation transcription sites. Nucleotide sequence analysis has revealed the absence of canonical TATA and CCAAT boxes and the presence of several putative regulatory elements in both promoter regions, including GAGA, GATA and Ets binding sites. We have analysed the pattern of gene expression during D. melanogaster development. The mRNA is stored in oocytes, and activation of transcription takes place after 10 h of development. The expression of the nuclear-encoded H(+)-ATP synthase beta subunit is strictly coordinated with the expression of subunits 6 and 8 of the same complex that are encoded in the mitochondrial genome. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:8554535

  3. Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers.

    PubMed

    Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K; Müller, Carsten T; Rosati, Carlo; Rogers, Hilary J

    2012-04-01

    Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar 'Sweet Laura' is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. 'Sweet Laura' with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. 'Sweet Laura' and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. 'Sweet Laura' placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R(28)(R)X(8)W and D(321)DXXD are the putative Mg(2+)-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. 'Sweet Laura' flowers.

  4. The ratios of aldosterone / plasma renin activity (ARR) versus aldosterone / direct renin concentration (ADRR).

    PubMed

    Glinicki, Piotr; Jeske, Wojciech; Bednarek-Papierska, Lucyna; Kruszyńska, Aleksandra; Gietka-Czernel, Małgorzata; Rosłonowska, Elżbieta; Słowińska-Srzednicka, Jadwiga; Kasperlik-Załuska, Anna; Zgliczyński, Wojciech

    2015-12-01

    Primary aldosteronism (PA) is estimated to occur in 5-12% of patients with hypertension. Assessment of aldosterone / plasma renin activity (PRA) ratio (ARR) has been used as a screening test in patients suspected of PA. Direct determination of renin (DRC) and calculation of aldosterone / direct renin concentration ratio (ADRR) could be similarly useful for screening patients suspected of PA. The study included 62 patients with indication for evaluation of the renin-angiotensin-aldosterone system and 35 healthy volunteers. In all participants we measured concentrations of serum aldosterone, plasma direct renin, and PRA after a night's rest and again after walking for two hours. The concentrations of aldosterone, direct renin, and PRA were measured by isotopic methods (radioimmunoassay (RIA) / immunoradiometric assay (IRMA)). Correlations of ARR with ADRR in the supine position were r = 0.9162, r(2) = 0.8165 (p < 0.01); and in the up-right position were r = 0.7765, r(2) = 0.9153 (p < 0.01). The cut-off values of ARR and ADRR ≥ 100 presented highest specificity (99%) for the diagnosis of PA; however, quite acceptable specificity and sensitivity (> 80% and 100%, respectively) appeared for the ratios ≥ 30. We suggest that for practical and economic reasons ARR can be replaced by ADRR.

  5. Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium.

    PubMed Central

    Bitzan, M M; Wang, Y; Lin, J; Marsden, P A

    1998-01-01

    Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E. coli O157-associated hemorrhagic colitis. We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-1 (ET-1) and nitric oxide (NO). In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h. Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis. In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged. Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-[14C]arginine to L-[14C]citrulline conversion or the NO chemiluminescence signal. The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations. Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells. Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis. Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the

  6. DHA Production in Escherichia coli by Expressing Reconstituted Key Genes of Polyketide Synthase Pathway from Marine Bacteria

    PubMed Central

    Xiao, Kang; Xu, Lin; Wang, Lian; Wan, Xia

    2016-01-01

    The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10–15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea. PMID:27649078

  7. Effects of quercetin on intracavernous pressure and expression of nitrogen synthase isoforms in arterial erectile dysfunction rat model

    PubMed Central

    Zhang, Yueyang; Huang, Changting; Liu, Shaoming; Bai, Jianqi; Fan, Xiaojing; Guo, Jun; Jia, Yingyu; Zhang, Zhijie; Chen, Xiaojun; Jia, Yusen; Zhang, Ping; Wang, Bin; Zhang, Xiuju

    2015-01-01

    Object: Oxidative stress involved in the regulation of arterial erectile dysfunction (A-ED). Previously report have indicated that quercetin have an antioxidant effect. In the current study, we have established the rats’ model for study the therapeutic effect of quercetin on A-ED and further investigated the molecular mechanism of action. Methods: Wistar rats were divided into sham group, A-ED group, A-ED group with low dose of quercetin, and A-ED group with high dose of quercetin. Intracavernous pressure (ICP) and mean arterial pressure (MBp) are two important indicators used for evaluation the A-ED. The changes of ICP and MBp were determined by cavernous nerve electrostimulation after treatment of quercetin at indicated doses. The expression of nitric oxide synthase (NOS) subtypes was detected by RT-PCR and Western blotting. Results: Our results indicated that ICP was significantly reduced in A-ED rats model compared with sham group, and was significantly increased after quercetin treatment (P < 0.01), while no significant effect on the MBp. The data also showed that sGC inhibitor ODQ and NOS inhibitor LNNA can significantly inhibited the ICP which induced by quercetin. These results suggest that NO-cGMP signaling pathway plays a crucial role in A-ED. Then, we found that the mRNA and protein levels of eNOS were significantly reduced in A-ED group compared with sham group. After treated with quercetin may cause the eNOS RNA and protein were significantly up-regulated (P < 0.01), showing a dose-dependent effect. iNOS expression have a certain degree of increased after quercetin treatment. nNOS expression was not significantly increased before and after treated with quercetin. In a word, quercetin can improved the A-ED by up-regulated ICP, which related to up-regulation of NO-cGMP signaling pathway. Conclusion: Preliminary results of this study suggested that quercetin protected expression and function of eNOS in cavernous endothelial cells, and restored part of

  8. Correlation of cytokines and inducible nitric oxide synthase expression with prognostic factors in ovarian cancer.

    PubMed

    Martins Filho, Agrimaldo; Jammal, Millena Prata; Côbo, Eliângela de Castro; Silveira, Thales Parenti; Adad, Sheila Jorge; Murta, Eddie Fernando Candido; Nomelini, Rosekeila Simões

    2014-01-01

    The study related the immunohistochemical staining of cytokines (IL2, IL5, IL6, IL8, IL10, and TNF-alpha), and iNOS staining with clinical and pathological parameters of patients with primary ovarian malignancy. We prospectively evaluated 40 patients who underwent surgical treatment in accordance with pre-established criteria and later confirmed diagnosis of ovarian cancer. Immunohistochemistry study for cytokines (IL2, IL5, IL6, IL8, IL10, TNF-alpha) and iNOS was performed. The evaluation of prognostic factors was performed using the Fisher's exact test. The significance level was less than 0.05. Histological grade 1 was significantly correlated with strong intensity for TNF-α (p=0.0028). In addition, early stages showed strong expression intensity of TNF-α, but this was at the limit of significance (p=0.0525). Strong staining immunohistochemical IL5 was related to disease-free survival less than or equal to 24 months, suggesting that a factor of poor prognosis, but there was no statistical significance (p=0.1771). There was no statistical significance in relation at other cytokines studied. Therefore, immunohistochemical staining in strong intensity for TNF-α was related to histological grade 1 and early stages of ovarian cancer in our sample of patients.

  9. High potassium promotes mutual interaction between (pro)renin receptor and the local renin-angiotensin-aldosterone system in rat inner medullary collecting duct cells.

    PubMed

    Xu, Chuanming; Fang, Hui; Zhou, Li; Lu, Aihua; Yang, Tianxin

    2016-10-01

    (Pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) with unclear functional implication. It is not known whether CD PRR is regulated by high potassium (HK). Here, we aimed to investigate the effect of HK on PRR expression and its role in regulation of aldosterone synthesis and release in the CD. In primary rat inner medullary CD cells, HK augmented PRR expression and soluble PPR (sPRR) release in a time- and dose-dependent manner, which was attenuated by PRR small interfering RNA (siRNA), eplerenone, and losartan. HK upregulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2) protein expression and upregulation of medium renin activity, both of which were attenuated by a PRR antagonist PRO20, PRR siRNA, eplerenone, and losartan. Similarly, prorenin upregulated aldosterone release and CYP11B2 expression, both of which were attenuated by PRR siRNA. Interestingly, a recombinant sPRR (sPRR-His) also stimulated aldosterone release and CYP11B2 expression. Taken together, we conclude that HK enhances a local renin-angiotensin-aldosterone system (RAAS), leading to increased PRR expression, which in turn amplifies the response of the RAAS, ultimately contributing to heightened aldosterone release.

  10. 4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase.

    PubMed

    Colby, S M; Alonso, W R; Katahira, E J; McGarvey, D J; Croteau, R

    1993-11-05

    The committed step in the biosynthesis of monoterpenes in mint (Mentha) species is the cyclization of geranyl pyrophosphate to the olefin (-)-4S-limonene catalyzed by limonene synthase (cyclase). Internal amino acid sequences of the purified enzyme from spearmint oil glands were utilized to design three distinct oligonucleotide probes. These probes were subsequently employed to screen a spearmint leaf cDNA library, and four clones were isolated. Three of these cDNA isolates were full-length and were functionally expressed in Escherichia coli, yielding a peptide that is immunologically recognized by polyclonal antibodies raised against the purified limonene synthase from spearmint and that is catalytically active in generating from geranyl pyrophosphate a product distribution identical to that of the native enzyme (principally limonene with small amounts of the coproducts alpha- and beta-pinene and myrcene). The longest open reading frame is 1800 nucleotides and the deduced amino acid sequence contains a putative plastidial transit peptide of approximately 90 amino acids and a mature protein of about 510 residues corresponding to the native enzyme. Several nucleotide differences in the 5'-untranslated region of all three full-length clones suggest the presence of several limonene synthase genes and/or alleles in the allotetraploid spearmint genome. Sequence comparisons with a sesquiterpene cyclase, epi-aristolochene synthase from tobacco, and a diterpene cyclase, casbene synthase from castor bean, demonstrated a significant degree of similarity between these three terpenoid cyclase types, the first three examples of this large family of catalysts to be described from higher plants.

  11. The acquisition of desiccation tolerance in developing Vicia hirsuta seeds coincides with an increase in galactinol synthase expression and soluble α-D-galactosides accumulation.

    PubMed

    Gojło, Ewa; Pupel, Piotr; Lahuta, Lesław B; Podliński, Paweł; Kucewicz, Magdalena; Górecki, Ryszard J

    2015-07-20

    Galactinol is the galactosyl donor for the biosynthesis of both the raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C). Its synthesis by galactinol synthase (GolS, EC 2.4.1.123) is the first committed step of the soluble α-D-galactosides biosynthetic pathway in orthodox seeds. The deposition of galactosides in seeds is suggested to be associated with desiccation tolerance (DT). In this work, for the first time, we cloned and characterized two Vicia hirsuta (L.) S.F. Gray galactinol synthase genes (VhGolS1, VhGolS2), analyzed galactinol synthase activity and measured the accumulation of galactosides of both sucrose and D-pinitol in relation to the acquisition of DT in developing seeds of this wild species. A developmentally induced increase of VhGolS1 expression preceded the rise of GolS activity in crude protein extract from maturing seeds, while the expression of the VhGolS2 gene remained low. GolS activity peaked just after the beginning of the maturation drying phase. The increase of GolS activity was not followed by galactinol accumulation, instead the high enzyme activity was related to high levels of galactose bound in soluble galactosides of the RFO and galactosyl pinitol series. Acquisition of DT coincided with an increase of VhGolS1 expression, high galactinol synthase activity and the accumulation of oligogalactosides in seeds. DT was positively correlated with the high content of soluble α-D-galactosides of both the RFO and galactosyl pinitol series as well as with the amount of galactose bound in these galactosides.

  12. Modulation of Immunity and Inflammation by the Mineralocorticoid Receptor and Aldosterone

    PubMed Central

    Muñoz-Durango, N.; Vecchiola, A.; Gonzalez-Gomez, L. M.; Simon, F.; Riedel, C. A.; Fardella, C. E.; Kalergis, A. M.

    2015-01-01

    The mineralocorticoid receptor (MR) is a ligand dependent transcription factor. MR has been traditionally associated with the control of water and electrolyte homeostasis in order to keep blood pressure through aldosterone activation. However, there is growing evidence indicating that MR expression is not restricted to vascular and renal tissues, as it can be also expressed by cells of the immune system, where it responds to stimulation or antagonism, controlling immune cell function. On the other hand, aldosterone also has been associated with proinflammatory immune effects, such as the release of proinflammatory cytokines, generating oxidative stress and inducing fibrosis. The inflammatory participation of MR and aldosterone in the cardiovascular disease suggests an association with alterations in the immune system. Hypertensive patients show higher levels of proinflammatory mediators that can be modulated by MR antagonism. Although these proinflammatory properties have been observed in other autoimmune and chronic inflammatory diseases, the cellular and molecular mechanisms that mediate these effects remain unknown. Here we review and discuss the scientific work aimed at determining the immunological role of MR and aldosterone in humans, as well as animal models. PMID:26448944

  13. Modulation of Immunity and Inflammation by the Mineralocorticoid Receptor and Aldosterone.

    PubMed

    Muñoz-Durango, N; Vecchiola, A; Gonzalez-Gomez, L M; Simon, F; Riedel, C A; Fardella, C E; Kalergis, A M

    2015-01-01

    The mineralocorticoid receptor (MR) is a ligand dependent transcription factor. MR has been traditionally associated with the control of water and electrolyte homeostasis in order to keep blood pressure through aldosterone activation. However, there is growing evidence indicating that MR expression is not restricted to vascular and renal tissues, as it can be also expressed by cells of the immune system, where it responds to stimulation or antagonism, controlling immune cell function. On the other hand, aldosterone also has been associated with proinflammatory immune effects, such as the release of proinflammatory cytokines, generating oxidative stress and inducing fibrosis. The inflammatory participation of MR and aldosterone in the cardiovascular disease suggests an association with alterations in the immune system. Hypertensive patients show higher levels of proinflammatory mediators that can be modulated by MR antagonism. Although these proinflammatory properties have been observed in other autoimmune and chronic inflammatory diseases, the cellular and molecular mechanisms that mediate these effects remain unknown. Here we review and discuss the scientific work aimed at determining the immunological role of MR and aldosterone in humans, as well as animal models.

  14. Genome-Wide Analysis of the Sucrose Synthase Gene Family in Grape (Vitis vinifera): Structure, Evolution, and Expression Profiles.

    PubMed

    Zhu, Xudong; Wang, Mengqi; Li, Xiaopeng; Jiu, Songtao; Wang, Chen; Fang, Jinggui

    2017-03-28

    Sucrose synthase (SS) is widely considered as the key enzyme involved in the plant sugar metabolism that is critical to plant growth and development, especially quality of the fruit. The members of SS gene family have been identified and characterized in multiple plant genomes. However, detailed information about this gene family is lacking in grapevine (Vitis vinifera L.). In this study, we performed a systematic analysis of the grape (V. vinifera) genome and reported that there are five SS genes (VvSS1-5) in the grape genome. Comparison of the structures of grape SS genes showed high structural conservation of grape SS genes, resulting from the selection pressures during the evolutionary process. The segmental duplication of grape SS genes contributed to this gene family expansion. The syntenic analyses between grape and soybean (Glycine max) demonstrated that these genes located in corresponding syntenic blocks arose before the divergence of grape and soybean. Phylogenetic analysis revealed distinct evolutionary paths for the grape SS genes. VvSS1/VvSS5, VvSS2/VvSS3 and VvSS4 originated from three ancient SS genes, which were generated by duplication events before the split of monocots and eudicots. Bioinformatics analysis of publicly available microarray data, which was validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct temporal and spatial expression patterns of VvSS genes in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. Taken together, our results will be beneficial for further investigations into the functions of SS gene in the processes of grape resistance to environmental stresses.

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

    PubMed

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

    2008-08-01

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

  16. Therapeutic perspectives in hypertension: novel means for renin-angiotensin-aldosterone system modulation and emerging device-based approaches.

    PubMed

    Unger, Thomas; Paulis, Ludovit; Sica, Domenic A

    2011-11-01

    The conventional antihypertensive therapies including renin-angiotensin-aldosterone system antagonists (converting enzyme inhibitors, receptor blockers, renin inhibitors, and mineralocorticoid receptor blockers), diuretics, β-blockers, and calcium channel blockers are variably successful in achieving the challenging target blood pressure values in hypertensive patients. Difficult to treat hypertension is still a commonly observed problem world-wide. A number of drugs are considered to be used as novel therapies for hypertension. Renalase supplementation, vasopeptidase inhibitors, endothelin antagonists, and especially aldosterone antagonists (aldosterone synthase inhibitors and novel selective mineralocorticoid receptor blockers) are considered an option in resistant hypertension. In addition, the aldosterone antagonists as well as (pro)renin receptor blockers or AT(2) receptor agonists might attenuate end-organ damage. This array of medications has now been complemented by a number of new approaches of non-pharmacological strategies including vaccination, genomic interference, controlled breathing, baroreflex activation, and probably most successfully renal denervation techniques. However, the progress on innovative therapies seems to be slow and the problem of resistant hypertension and proper blood pressure control appears to be still persisting. Therefore the regimens of currently available drugs are being fine-tuned, resulting in the establishment of several novel fixed-dose combinations including triple combinations with the aim to facilitate proper blood pressure control. It remains an exciting question which approach will confer the best blood pressure control and risk reduction in this tricky disease.

  17. The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria

    SciTech Connect

    Silvester, Jocelyn A.; Kane Dickson, Veronica; Runswick, Michael J.; Leslie, Andrew G. W.; Walker, John E.

    2006-06-01

    A recombinant subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been crystallized and a native data set has been collected to 2.8 Å resolution. A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79–184 of subunit b, residues 1–124 of subunit d and the entire F{sub 6} subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 Å resolution. They belong to the monoclinic space group P2{sub 1}.

  18. A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

    PubMed

    Formighieri, Cinzia; Melis, Anastasios

    2015-11-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.

  19. Sensitivity of Aspergillus nidulans to the cellulose synthase inhibitor dichlobenil: insights from wall-related genes' expression and ultrastructural hyphal morphologies.

    PubMed

    Guerriero, Gea; Silvestrini, Lucia; Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

    2013-01-01

    The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes' expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.

  20. Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

    PubMed Central

    Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

    2013-01-01

    The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

  1. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

    PubMed Central

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M.; Baerga-Ortiz, Abel

    2014-01-01

    Increasing the production of fatty acids by microbial fermentation remains an important step towards the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations towards accessible biodiesel precursors. PMID:24411456

  2. Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2{Delta} mutant and CHO cells.

    PubMed

    Rush, Jeffrey S; Matveev, Sergey; Guan, Ziqiang; Raetz, Christian R H; Waechter, C J

    2010-12-01

    During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc(3)Man(9)GlcNAc(2)-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and glycosylphosphatidylinositol anchor assembly. In support of this conclusion, mutant cells expressing UPPS (1) synthesized (55)Dol-P based on MS analysis, (2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo, and (3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [(3)H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the endoplasmic reticulum of Chinese hamster ovary cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [(14)C](55)polyprenyl-P-P intermediate when incubated with [(14)C]isopentenyl pyrophosphate and [(3)H]Man-P-(55)Dol when incubated with GDP-[(3)H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the endoplasmic reticulum of yeast and mammalian cells, and that conserved sequences in the cis

  3. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli.

    PubMed

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M; Baerga-Ortiz, Abel

    2014-02-05

    Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.

  4. Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

    PubMed Central

    Victorio, Jamaira A.; Clerici, Stefano P.; Palacios, Roberto; Alonso, María J.; Vassallo, Dalton V.; Jaffe, Iris Z.; Rossoni, Luciana V.

    2016-01-01

    Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation. PMID:27432866

  5. Upregulation of Steroidogenic Acute Regulatory Protein by Hypoxia Stimulates Aldosterone Synthesis in Pulmonary Artery Endothelial Cells to Promote Pulmonary Vascular Fibrosis

    PubMed Central

    Maron, Bradley A.; Oldham, William M.; Chan, Stephen Y.; Vargas, Sara O.; Arons, Elena; Zhang, Ying-Yi; Loscalzo, Joseph; Leopold, Jane A.

    2014-01-01

    Background The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in PAH. Methods and Results Patients with PAH, rats with Sugen/hypoxia-PAH, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia-StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography-mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein (AP-1) site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR-siRNA or treated with the AP-1 inhibitor, SR-11302, hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro, and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in Conclusions Our findings identify autonomous aldosterone synthesis in HPAECs due to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular

  6. Factors affecting the aldosterone/renin ratio.

    PubMed

    Stowasser, M; Ahmed, A H; Pimenta, E; Taylor, P J; Gordon, R D

    2012-03-01

    Although the aldosterone/renin ratio (ARR) is the most reliable screening test for primary aldo-steronism, false positives and negatives occur. Dietary salt restriction, concomitant malignant or renovascular hypertension, pregnancy and treatment with diuretics (including spironolactone), dihydropyridine calcium blockers, angiotensin converting enzyme inhibitors, and angiotensin receptor antagonists can produce false negatives by stimulating renin. We recently reported selective serotonin reuptake inhibitors lower the ratio. Because potassium regulates aldosterone, uncorrected hypokalemia can lead to false negatives. Beta-blockers, alpha-methyldopa, clonidine, and nonsteroidal anti-inflammatory drugs suppress renin, raising the ARR with potential for false positives. False positives may occur in patients with renal dysfunction or advancing age. We recently showed that (1) females have higher ratios than males, and (2) false positive ratios can occur during the luteal menstrual phase and while taking an oral ethynylestradiol/drospirenone (but not implanted subdermal etonogestrel) contraceptive, but only if calculated using direct renin concentration and not plasma renin activity. Where feasible, diuretics should be ceased at least 6 weeks and other interfering medications at least 2 before ARR measurement, substituting noninterfering agents (e. g., verapamil slow-release±hydralazine and prazosin or doxazosin) were required. Hypokalemia should be corrected and a liberal salt diet encouraged. Collecting blood midmorning from seated patients following 2-4 h upright posture improves sensitivity. The ARR is a screening test only and should be repeated once or more before deciding whether to proceed to confirmatory suppression testing. Liquid chromatography-tandem mass spectrometry aldosterone assays represent a major advance towards addressing inaccuracies inherent in other available methods.

  7. Moderate inappropriately high aldosterone/NaCl constellation in mice: cardiovascular effects and the role of cardiovascular epidermal growth factor receptor.

    PubMed

    Schreier, Barbara; Rabe, Sindy; Winter, Sabrina; Ruhs, Stefanie; Mildenberger, Sigrid; Schneider, Bettina; Sibilia, Maria; Gotthardt, Michael; Kempe, Sabine; Mäder, Karsten; Grossmann, Claudia; Gekle, Michael

    2014-12-11

    Non-physiological activation of the mineralocorticoid receptor (MR), e.g. by aldosterone under conditions of high salt intake, contributes to the pathogenesis of cardiovascular diseases, although beneficial effects of aldosterone also have been described. The epidermal growth factor receptor (EGFR) contributes to cardiovascular alterations and mediates part of the MR effects. Recently, we showed that EGFR is required for physiological homeostasis and function of heart and arteries in adult animals. We hypothesize that moderate high aldosterone/NaCl, at normal blood pressure, affects the cardiovascular system depending on cardiovascular EGFR. Therefore we performed an experimental series in male and female animals each, using a recently established mouse model with EGFR knockout in vascular smooth muscle cells and cardiomyocytes and determined the effects of a mild-high aldosterone-to-NaCl constellation on a.o. marker gene expression, heart size, systolic blood pressure, impulse conduction and heart rate. Our data show that (i) cardiac tissue of male but not of female mice is sensitive to mild aldosterone/NaCl treatment, (ii) EGFR knockout induces stronger cardiac disturbances in male as compared to female animals and (iii) mild aldosterone/NaCl treatment requires the EGFR in order to disturb cardiac tissue homeostasis whereas beneficial effects of aldosterone seem to be independent of EGFR.

  8. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-01-01

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

  9. Molecular cloning, characterization and mRNA expression of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

    PubMed

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-08-19

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

  10. Molecular cloning and expression profile of ß-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a ...

  11. Albuminuria is associated with an increased prostasin in urine while aldosterone has no direct effect on urine and kidney tissue abundance of prostasin.

    PubMed

    Oxlund, Christina; Kurt, Birgül; Schwarzensteiner, Ilona; Hansen, Mie R; Stæhr, Mette; Svenningsen, Per; Jacobsen, Ib A; Hansen, Pernille B; Thuesen, Anne D; Toft, Anja; Hinrichs, Gitte R; Bistrup, Claus; Jensen, Boye L

    2017-02-24

    The proteinase prostasin is a candidate mediator for aldosterone-driven proteolytic activation of the epithelial sodium channel (ENaC). It was hypothesized that the aldosterone-mineralocorticoid receptor (MR) pathway stimulates prostasin abundance in kidney and urine. Prostasin was measured in plasma and urine from type 2 diabetic patients with resistant hypertension (n = 112) randomized to spironolactone/placebo in a clinical trial. Prostasin protein level was assessed by immunoblotting in (1) human and rat urines with/without nephrotic syndrome, (2) human nephrectomy tissue, (3) urine and kidney from aldosterone synthase-deficient (AS(-/-)) mice and ANGII- and aldosterone-infused mice, and in (4) kidney from adrenalectomized rats. Serum aldosterone concentration related to prostasin concentration in urine but not in plasma. Plasma prostasin concentration increased significantly after spironolactone compared to control. Urinary prostasin and albumin related directly and were reduced by spironolactone. In patients with nephrotic syndrome, urinary prostasin protein was elevated compared to controls. In rat nephrosis, proteinuria coincided with increased urinary prostasin, unchanged kidney tissue prostasin, and decreased plasma prostasin while plasma aldosterone was suppressed. Prostasin protein abundance in human nephrectomy tissue was similar across gender and ANGII inhibition regimens. Prostasin urine abundance was not different in AS(-/-) and aldosterone-infused mice. Prostasin kidney level was not different from control in adrenalectomized rats and AS(-/-) mice. We found no evidence for a direct relationship between mineralocorticoid receptor signaling and kidney and urine prostasin abundance. The reduction of urinary prostasin in spironolactone-treated patients is most likely the result of an improved glomerular filtration barrier function and generally reduced proteinuria.

  12. Molecular cloning and differential expressions of two cDNA encoding Type III polyketide synthase in different tissues of Curcuma longa L.

    PubMed

    Resmi, M S; Soniya, E V

    2012-01-10

    Type III polyketide synthase family of enzymes play an important role in the biosynthesis of flavonoids and a variety of plant polyphenols by condensing multiple acetyl units derived from malonyl Co-A to thioester linked starter molecules covalently bound in the PKS active site. Turmeric (Curucma longa L.) through diverse metabolic pathways produces a large number of metabolites, of which curcuminoids had gained much attention due to its immense pharmaceutical value. Recent identification of multiple curcuminoid synthases from turmeric lead us to look for additional Type III PKS from this plant. The current study describes the occurrence of a multigene family of Type III PKS enzymes in C. longa by RT-PCR based genomic screening. We have also isolated two new Type III PKS, ClPKS9 and ClPKS10 using homology based RT-PCR and data mining. The comparative sequence and phylogenetic analysis revealed that the two PKSs belong to different groups with only 56% sequence similarity at their amino acid level. ClPKS9 shows all possible sequence requirements for a typical chalcone synthase whereas ClPKS10 shows promising variation at amino acid level and high similarity to reported curcuminoid synthases. ClPKS9 and ClPKS10 exhibited distinct tissue specific expression pattern in C. longa with the ClPKS9 transcript abundant in shoot and rhizome than leaves whereas ClPKS10 transcript was found to be high in leaf and very low in rhizome and root. Therefore it was concluded that ClPKS9 and ClPKS10 may have divergent function in planta, with possible role in typical chalcone forming reaction and curcuminoid scaffold biosynthetic pathway respectively.

  13. Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis.

    PubMed

    Colón, A L; Menchén, L A; Hurtado, O; De Cristóbal, J; Lizasoain, I; Leza, J C; Lorenzo, P; Moro, M A

    2001-12-21

    Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.

  14. Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers

    PubMed Central

    Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K.; Müller, Carsten T.; Rosati, Carlo; Rogers, Hilary J.

    2012-01-01

    Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar ‘Sweet Laura’ is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. ‘Sweet Laura’ with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. ‘Sweet Laura’ and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. ‘Sweet Laura’ placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R28(R)X8W and D321DXXD are the putative Mg2+-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. ‘Sweet Laura’ flowers. PMID:22268153

  15. Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’ 1[W][OA

    PubMed Central

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

    2012-01-01

    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

  16. Evaluation of insulin sensitivity and secretion in primary aldosteronism.

    PubMed

    Watanabe, Daisuke; Yatabe, Midori; Ichihara, Atsuhiro

    In primary aldosteronism (PA), insulin response to glucose is not fully understood. Insulin action was elucidated using indices in 32 PA and 21 essential hypertension (EH) patients. These patients were evaluated using homeostasis model assessment (HOMA) indices, quantitative insulin sensitivity check index (QUICKI), and insulinogenic index (IGI), which were expressed for insulin sensitivity/secretion and the early phase of insulin secretion. Insulin sensitivity and early phase of insulin secretion were decreased in PA, and there was a negative correlation between serum potassium concentration and insulin sensitivity indices. These findings suggest that glucose intolerance in PA may be caused by hypokalemia-induced insulin resistance and hypokalemia-independent impairment of early-phase insulin secretion.

  17. Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane.

    PubMed

    Hamerli, Dénes; Birch, Robert G

    2011-01-01

    Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole-targeted trehalulose synthase modified from the gene in 'Pseudomonas mesoacidophila MX-45' and controlled by the maize ubiquitin (Ubi-1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mM, with near-complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.

  18. Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway.

    PubMed

    Ren, Ang; Ouyang, Xiang; Shi, Liang; Jiang, Ai-Liang; Mu, Da-Shuai; Li, Meng-Jiao; Han, Qin; Zhao, Ming-Wen

    2013-03-01

    A hydroxymethylglutaryl-CoA synthase gene, designated as GlHMGS (GenBank accession No. JN391469) involved in ganoderic acid (GA) biosynthesis pathway was cloned from Ganoderma lucidum. The full-length cDNA of GlHMGS (GenBank accession No. JN391468) was found to contain an open reading frame of 1,413 bp encoding a polypeptide of 471 amino acid residues. The deduced amino acid sequence of GlHMGS shared high homology with other known hydroxymethylglutaryl-CoA synthase (HMGS) enzymes. In addition, functional complementation of GlHMGS in a mutant yeast strain YSC1021 lacking HMGS activity demonstrated that the cloned cDNA encodes a functional HMGS. A 1,561 bp promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. GlHMGS expression profile analysis revealed that salicylic acid, abscisic acid and methyl jasmonate up-regulated GlHMGS transcript levels over the control. Further expression analysis revealed that the developmental stage and carbon source had significant effects on GlHMGS transcript levels. GlHMGS expression peaked on day 16 before decreasing with prolonged culture time. The highest mRNA level was observed when the carbon source was maltose. Overexpression of GlHMGS enhanced GA content in G. lucidum. This study provides useful information for further studying this gene and on its function in the ganoderic acid biosynthetic pathway in G. lucidum.

  19. Callose and cellulose synthase gene expression analysis from the tight cluster to the full bloom stage and during early fruit development in Malus × domestica.

    PubMed

    Guerriero, Gea; Giorno, Filomena; Folgado, Raquel; Printz, Bruno; Baric, Sanja; Hausman, Jean-Francois

    2014-01-01

    Apple (Malus × domestica) is an economically important temperate fruit-bearing crop which belongs to the family of Rosaceae and its pomaceous fruit is one of the most commonly cultivated. Several studies have demonstrated that the cell wall plays a pivotal role during flower and fruit development. It takes active part in pollen tube growth and contributes to determine the fruit firmness trait through the action of cell wall-related enzymes (i.e. polygalacturonase and pectinmethylesterase). We have investigated the expression of callose and cellulose synthase genes during flowering from tight cluster to anthesis and during early fruit development in domesticated apple. We also link the changes observed in gene expression to the profile of soluble non-structural carbohydrates at different developmental stages of flowers/fruitlets and to the qualitative results linked to wall polysaccharides' composition obtained through near-infrared spectroscopy. This work represents an important addition to the study of tree physiology with respect to the analysis of the expression of callose and cellulose synthase genes during flower and early fruit development in domesticated apple.

  20. Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2013-10-01

    Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

  1. Leptin Induces Hypertension and Endothelial Dysfunction via Aldosterone-Dependent Mechanisms in Obese Female Mice.

    PubMed

    Huby, Anne-Cécile; Otvos, Laszlo; Belin de Chantemèle, Eric J

    2016-05-01

    Obesity is a major risk factor for cardiovascular disease in males and females. Whether obesity triggers cardiovascular disease via similar mechanisms in both the sexes is, however, unknown. In males, the adipokine leptin highly contributes to obesity-related cardiovascular disease by increasing sympathetic activity. Females secrete 3× to 4× more leptin than males, but do not exhibit high sympathetic tone with obesity. Nevertheless, females show inappropriately high aldosterone levels that positively correlate with adiposity and blood pressure (BP). We hypothesized that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in females. Leptin control of the cardiovascular function was analyzed in female mice sensitized to leptin via the deletion of protein tyrosine phosphatase 1b (knockout) and in agouti yellow obese hyperleptinemic mice (Ay). Hypersensitivity to leptin (wild-type, 115 ± 2; protein tyrosine phosphatase 1b knockout, 124 ± 2 mm Hg; P<0.05) and obesity elevated BP (a/a, 113 ± 1; Ay, 128 ± 7 mm Hg; P<0.05) and impaired endothelial function. Chronic leptin receptor antagonism restored BP and endothelial function in protein tyrosine phosphatase 1b knockout and Ay mice. Hypersensitivity to leptin and obesity reduced BP response to ganglionic blockade in both strains and plasma catecholamine levels in protein tyrosine phosphatase 1b knockout mice. Hypersensitivity to leptin and obesity significantly increased plasma aldosterone levels and adrenal CYP11B2 expression. Chronic leptin receptor antagonism reduced aldosterone levels. Furthermore, chronic leptin and mineralocorticoid receptor blockade reduced BP and improved endothelial function in both leptin-sensitized and obese hyperleptinemic female mice. Together, these data demonstrate that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in female mice and suggest that obesity leads to cardiovascular disease via sex

  2. Leptin Induces Hypertension and Endothelial Dysfunction via Aldosterone-Dependent Mechanisms in Obese Female Mice

    PubMed Central

    Huby, Anne-Cecile; Otvos, Laszlo; Belin de Chantemèle, Eric J.

    2016-01-01

    Obesity is a major risk factor for cardiovascular disease in males and females. Whether obesity triggers cardiovascular disease via similar mechanisms in both the sexes is, however, unknown. In males, the adipokine leptin highly contributes to obesity-related cardiovascular disease by increasing sympathetic activity. Females secrete 3× to 4× more leptin than males, but do not exhibit high sympathetic tone with obesity. Nevertheless, females show inappropriately high aldosterone levels that positively correlate with adiposity and blood pressure (BP). We hypothesized that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in females. Leptin control of the cardiovascular function was analyzed in female mice sensitized to leptin via the deletion of protein tyrosine phosphatase 1b (knockout) and in agouti yellow obese hyperleptinemic mice (Ay). Hypersensitivity to leptin (wild-type, 115±2; protein tyrosine phosphatase 1b knockout, 124±2 mm Hg; P<0.05) and obesity elevated BP (a/a, 113±1; Ay, 128±7 mm Hg; P<0.05) and impaired endothelial function. Chronic leptin receptor antagonism restored BP and endothelial function in protein tyrosine phosphatase 1b knockout and Ay mice. Hypersensitivity to leptin and obesity reduced BP response to ganglionic blockade in both strains and plasma catecholamine levels in protein tyrosine phosphatase 1b knockout mice. Hypersensitivity to leptin and obesity significantly increased plasma aldosterone levels and adrenal CYP11B2 expression. Chronic leptin receptor antagonism reduced aldosterone levels. Furthermore, chronic leptin and mineralocorticoid receptor blockade reduced BP and improved endothelial function in both leptin-sensitized and obese hyperleptinemic female mice. Together, these data demonstrate that leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in female mice and suggest that obesity leads to cardiovascular disease via sex

  3. Aldosterone Inhibits the Fetal Program and Increases Hypertrophy in the Heart of Hypertensive Mice

    PubMed Central

    Azibani, Feriel; Devaux, Yvan; Coutance, Guillaume; Schlossarek, Saskia; Polidano, Evelyne; Fazal, Loubina; Merval, Regine; Carrier, Lucie; Solal, Alain Cohen; Chatziantoniou, Christos; Launay, Jean-Marie; Samuel, Jane-Lise; Delcayre, Claude

    2012-01-01

    Background Arterial hypertension (AH) induces cardiac hypertrophy and reactivation of “fetal” gene expression. In rodent heart, alpha-Myosin Heavy Chain (MyHC) and its micro-RNA miR-208a regulate the expression of beta-MyHC and of its intronic miR-208b. However, the role of aldosterone in these processes remains unclear. Methodology/Principal Findings RT-PCR and western-blot were used to investigate the genes modulated by arterial hypertension and cardiac hyperaldosteronism. We developed a model of double-transgenic mice (AS-Ren) with cardiac hyperaldosteronism (AS mice) and systemic hypertension (Ren). AS-Ren mice had increased (x2) angiotensin II in plasma and increased (x2) aldosterone in heart. Ren and AS-Ren mice had a robust and similar hypertension (+70%) versus their controls. Anatomical data and echocardiography showed a worsening of cardiac hypertrophy (+41%) in AS-Ren mice (P<0.05 vs Ren). The increase of ANP (x 2.5; P<0.01) mRNA observed in Ren mice was blunted in AS-Ren mice. This non-induction of antitrophic natriuretic peptides may be involved in the higher trophic cardiac response in AS-Ren mice, as indicated by the markedly reduced cardiac hypertrophy in ANP-infused AS-Ren mice for one month. Besides, the AH-induced increase of ßMyHC and its intronic miRNA-208b was prevented in AS-Ren. The inhibition of miR 208a (−75%, p<0.001) in AS-Ren mice compared to AS was associated with increased Sox 6 mRNA (x 1.34; p<0.05), an inhibitor of ßMyHC transcription. Eplerenone prevented all aldosterone-dependent effects. Conclusions/Significance Our results indicate that increased aldosterone in heart inhibits the induction of atrial natriuretic peptide expression, via the mineralocorticoid receptor. This worsens cardiac hypertrophy without changing blood pressure. Moreover, this work reveals an original aldosterone-dependent inhibition of miR-208a in hypertension, resulting in the inhibition of β-myosin heavy chain expression through the induction of

  4. The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells.

    PubMed

    Pan, Zhi-qiang; Xie, Ding; Choudhary, Vivek; Seremwe, Mutsa; Tsai, Ying-Ying; Olala, Lawrence; Chen, Xunsheng; Bollag, Wendy B

    2014-08-25

    Pioglitazone belongs to the class of drugs called thiazolidinediones (TZDs), which are widely used as insulin sensitizers in the treatment of diabetes. A major side effect of TZDs is fluid retention. The steroid hormone aldosterone also promotes sodium and fluid retention; however, the effect of pioglitazone on aldosterone production is controversial. We analyzed the effect of pioglitazone alone and in combination with angiotensin II (AngII) on the late rate-limiting step of adrenocortical steroidogenesis in human adrenocortical carcinoma HAC15 cells. Treatment with pioglitazone for 24 h significantly increased the expression of CYP11B2 and enhanced AngII-induced CYP11B2 expression. Despite the observed changes in mRNA levels, pioglitazone significantly inhibited AngII-induced aldosterone production and CYP11B2 protein levels. On the other hand, pioglitazone stimulated the expression of the unfolded protein response (UPR) marker DDIT3, with this effect occurring at early times and inhibitable by the PPARγ antagonist GW9962. The levels of DDIT3 (CHOP) and phospho-eIF2α (Ser51), a UPR-induced event that inhibits protein translation, were also increased. Thus, pioglitazone promotes CYP11B2 expression but nevertheless inhibits aldosterone production in AngII-treated HAC15 cells, likely by blocking global protein translation initiation through DDIT3 and phospho-eIF2α. In contrast, pioglitazone promoted AngII-induced CYP11B1 expression and cortisol production. Since cortisol enhances lipolysis, this result suggests the possibility that PPARs, activated by products of fatty acid oxidation, stimulate cortisol secretion to promote utilization of fatty acids during fasting. In turn, the ability of pioglitazone to stimulate cortisol production could potentially underlie the effects of this drug on fluid retention.

  5. Prognostic significance of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in biliary tract cancer patients receiving adjuvant 5-fluorouracil-based chemotherapy

    PubMed Central

    KIM, KWAN WOO; KWON, HYUK-CHAN; KIM, SUNG-HYUN; OH, SUNG YONG; LEE, SUEE; LEE, JI HYUN; ROH, MYUNG HWAN; KIM, MIN CHAN; KIM, KI HAN; KIM, YOUNG HOON; ROH, YOUNG HOON; JEONG, JIN SOOK; KIM, HYO-JIN

    2013-01-01

    Biliary tract cancer (BTC) is a relatively uncommon type of cancer, accounting for ∼4% of the malignant neoplasms of the gastrointestinal tract. The aim of this study was to determine whether the expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) predict clinical outcome in BTC patients treated with adjuvant 5-fluorouracil (5-FU)-based chemotherapy. TS and TP expression were found to be significantly correlated with cancer location (P=0.044 and 0.031, respectively). The multivariate analysis revealed that age [hazard ratio (HR)=2.157, P=0.008], stage (HR=2.234, P<0.001), resection margin status (HR=2.748, P=0.004) and TP expression (HR=2.014, P=0.039) were independently associated with overall survival (OS). PMID:24649282

  6. The Regulation of Aldosterone Secretion in Anephric Man

    PubMed Central

    Bayard, Francis; Cooke, C. Robert; Tiller, David J.; Beitins, Inese Z.; Kowarski, Avinoam; Walker, W. Gordon; Migeon, Claude J.

    1971-01-01

    The regulation of aldosterone secretion in anephric man was investigated in studies on nephrectomized patients who were being intermittently hemodialyzed while awaiting renal transplantation. The effects of supine and upright posture on the concentration of plasma aldosterone on the 1st day postdialysis and on a 3rd or 4th day postdialysis were compared to the effects of postural variation in normal subjects who were on a low sodium intake and on a high sodium intake. In contrast with the normal subjects who exhibited higher concentrations of plasma aldosterone after 2 hr of upright posture than in the supine position and low concentrations of plasma aldosterone on a high sodium intake, the anephric patients showed less consistent variations in plasma aldosterone due to changes in posture and exhibited higher concentrations of plasma aldosterone on the 3rd or 4th day postdialysis, despite an increase in body weight, than on the 1st day postdialysis. The increase in the concentration of plasma aldosterone in the anephric patients between the 1st day postdialysis and the 3rd or 4th day postdialysis indicates that aldosterone secretion is not responding primarily, in this situation, to volume-related stimuli. There was a high degree of correlation between the concentration of plasma aldosterone and the corresponding levels of serum potassium concentration, which also rose significantly between the 1st day postdialysis and the 3rd or 4th day postdialysis. Furthermore, when potassium accumulation between dialyses was prevented in three of these patients, the concentration of plasma aldosterone fell to minimally detectable levels. The results of these studies suggest that the primary regulator of aldosterone secretion in the absence of the kidneys is potassium. PMID:4329001

  7. Alteration of amiloride-sensitive salt taste nerve responses in aldosterone/NaCl-induced hypertensive rats.

    PubMed

    Sakamoto, Takashi; Fujii, Akihiko; Saito, Naoko; Kondo, Hidehiko; Ohuchi, Atsushi

    2016-07-01

    Salt taste sensitivity is related to physiological condition, and declined in hypertensive patients. However, little is known about the mechanism underlying changes in salt taste sensitivity during the development of hypertension. This is largely due to lack of an appropriate animal model which shows the decline of salt taste sensitivity caused by hypertension. Previous studies have suggested that one of main causes of salt-sensitive hypertension is dysfunction of the renin-angiotensin-aldosterone system (RAAS). To examine the involvement of RAAS in modulation of salt taste sensitivity, we utilized aldosterone/NaCl-treated rats as a well-established model of salt-sensitive hypertension caused by RAAS dysfunction. Amount of sodium intake in aldosterone/NaCl-treated rats was higher than that in control rats. In addition to behavioral changes, the amiloride-sensitive salt taste nerve responses in aldosterone/NaCl-treated rats were remarkably lower by approximately 90% than those in the other groups. Moreover, αENaC mRNA expression in the epithelium of circumvallate papillae was significantly low in aldosterone/NaCl-treated rats. Thus, RAAS modulates salt taste system as is case in hypertensive patients. This report is to our knowledge the first to describe an animal model with decline of amiloride-sensitive salt taste nerve responses by RAAS dysfunction-mediated salt-sensitive hypertension.

  8. The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon

    PubMed Central

    Wang, Xiao-Yan; Masilamani, Shyama; Nielsen, Jakob; Kwon, Tae-Hwan; Brooks, Heddwen L.; Nielsen, Søren; Knepper, Mark A.

    2001-01-01

    The kidneys “escape” from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism. J. Clin. Invest. 108:215–222 (2001). DOI:10.1172/JCI200110366. PMID:11457874

  9. Inhibition of brain oxidative stress and inducible nitric oxide synthase expression by thymoquinone attenuates the development of morphine tolerance and dependence in mice.

    PubMed

    Abdel-Zaher, Ahmed O; Mostafa, Mostafa G; Farghly, Hanan M; Hamdy, Mostafa M; Omran, Ghada A; Al-Shaibani, Najlaà K M

    2013-02-28

    In this study, the effect of thymoquinone on morphine-induced tolerance and dependence in mice was investigated. Repeated administration of thymoquinone along with morphine attenuated the development of morphine tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone-precipitated withdrawal manifestations. Concurrently, morphine-induced progressive increase in brain malondialdehyde (MDA) level and nitric oxide (NO) production as well as progressive decrease in brain intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity were inhibited by co-administration of thymoquinone. Morphine-induced progressive increase in brain glutamate level was not inhibited by concomitant administration of thymoquinone. Similarly, co-administration of thymoquinone inhibited naloxone-induced increase in brain MDA level, NO overproduction and decrease in brain intracellular GSH level and GSH-Px activities but it did not inhibit naloxone-induced elevation of brain glutamate level in morphine-dependent mice. The inhibitory effect of thymoquinone on morphine-induced tolerance and dependence on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine, the antioxidant, N-acetylcysteine or the NO synthase inhibitor, L-N (G)-nitroarginine methyl ester. On the other hand, this inhibitory effect of thymoquinone was antagonized by concurrent i.p. administration of NO precursor, L-arginine. In addition, concomitant administration of thymoquinone inhibited morphine tolerance and dependence-induced increase in inducible but not in neuronal NO synthase mRNA expression in mice brain. These results demonstrate that inhibition of morphine-induced oxidative stress, increase in the expression of brain inducible NO synthase and NO overproduction by thymoquinone can attenuate the development of morphine tolerance and dependence.

  10. Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids.

    PubMed

    Lan, Jian-bin; Yu, Rang-cai; Yu, Yun-yi; Fan, Yan-ping

    2013-04-15

    Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.

  11. Effect of N-nitrosodimethylamine on inducible nitric oxide synthase expression and production of nitric oxide by neutrophils and mononuclear cells: the role of JNK signalling pathway.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

    2011-07-01

    In neutrophils (PMN) and mononuclear cells (PBMC), one of the enzymes responsible for nitric oxide (NO) synthesis is inducible nitric oxide synthase (iNOS). Changes in iNOS expression result from various signalling pathways including the mitogen-activated protein kinase (MAPK) pathway activated by endogenic and exogenic factors. N-nitrosodimethylamine (NDMA) is a xenobiotic with widespread occurrence in human environment and has an effect on cells of the immune system. The aim of this study was to determine the effect of NDMA on iNOS expression and NO production by human PMN and PBMC cells in the light of superoxide anion production by PMN cells. Moreover, the role of JNK and p38 pathways in NO production with involvement of iNOS was studied. Additionally, the function of JNK pathway in generation of superoxide anion was determined. Moreover, nitrotyrosine expression was studied in PMN and PBMC cells in the presence of NDMA. This work shows that NDMA increases iNOS expression and NO production by PMN and PBMC cells. In addition, elevated expression of phospho-JNK and phospho-p38, which are markers of JNK and p38 MAPK pathways activation, were observed. Lower iNOS expression and NO production by neutrophils exposed to extended action of NDMA were observed after application of inhibitor of JNK and p38 pathways. Lower phospho-p38 expression in PMN stimulated by NDMA was found as a result of arresting JNK pathway, whereas, application of inhibitor of p38 pathway resulted in enhanced phospho-JNK expression in PMN and PBMC cells. Increased ability to release superoxide anion by NDMA-stimulated PMN cells was observed. This ability was reduced after the application of inhibitor of JNK pathway. In PMN and PBMC cells exposed to NDMA, an increased expression of nitrotyrosine, which is dependant on JNK and p38 pathways that are activated by this particular xenobiotic, was observed. Generally, increased induction of iNOS related to elevated production of NO by PMN and PBMC

  12. Hyperkalaemia in the age of aldosterone antagonism.

    PubMed

    Chapagain, A; Ashman, N

    2012-11-01

    Hyperkalaemia is well recognized as a medical emergency. However, with the publication of trials showing benefit with renin-aldosterone axis suppression in heart failure, the epidemiology of patients presenting with hyperkalaemia has changed. The reported incidence of rate of serious hyperkalaemia (>6.0 mEq/l of potassium) ranges from 6 to 12% in patients on spironolactone with congestive cardiac failure (CCF). A rational choice of therapy based on present evidence is different from the traditionally used algorithm, given our understanding of the physiology relevant to this patient group. This article discusses the changing face of hyperkalaemia and the present evidence and discusses options in treatment of hyperkalaemia.

  13. T-shaped trichome-specific expression of monoterpene synthase ADH2 using promoter-β-GUS fusion in transgenic Artemisia annua L.

    PubMed

    Fu, Xueqing; Shi, Pu; Shen, Qian; Jiang, Weimin; Tang, Yueli; Lv, Zongyou; Yan, Tingxiang; Li, Ling; Wang, Guofeng; Sun, Xiaofen; Tang, Kexuan

    2016-11-01

    Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (sweet wormwood), is extensively used in the treatment of malaria. In order to better understand the metabolism of terpenes in A. annua and the influence of terpene synthases on artemisinin yield, the expression pattern of a monoterpene alcohol dehydrogenase (ADH2) has been studied using transgenic plants expressing promoter-β-glucuronidase (GUS) fusion. ADH2 played a major role in monoterpenoid biosynthesis including carveol, borneol, and artemisia ketone through in vitro biochemical analysis. In this study, the ADH2 promoter was cloned by the genome walking method. A number of putative cis-acting elements were predicted in promoter region, suggesting that the ADH2 is driven by a complex regulation mechanism. ADH2 gene was highly expressed in old leaves, whereas the artemisinin biosynthetic genes were mainly expressed in bud and young leaves. The expression of ADH2 gene increased quickly during leaf development, revealed by qRT-PCR. GUS expression analysis in different tissues of transgenic A. annua demonstrates that ADH2 expression is exclusively located to T-shaped trichome, not glandular secretory trichome.

  14. Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators.

    PubMed Central

    Guo, F H; Uetani, K; Haque, S J; Williams, B R; Dweik, R A; Thunnissen, F B; Calhoun, W; Erzurum, S C

    1997-01-01

    Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known. We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA. IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk. Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC. IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not. Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation. In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA. PMID:9259582

  15. DACH1, a zona glomerulosa selective gene in the human adrenal, activates transforming growth factor-β signaling and suppresses aldosterone secretion.

    PubMed

    Zhou, Junhua; Shaikh, Lalarukh Haris; Neogi, Sudeshna G; McFarlane, Ian; Zhao, Wanfeng; Figg, Nichola; Brighton, Cheryl A; Maniero, Carmela; Teo, Ada E D; Azizan, Elena A B; Brown, Morris J

    2015-05-01

    Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-β, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-β and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-β signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.

  16. Aldosterone and mineralocorticoid receptors in the cardiovascular system.

    PubMed

    Funder, John W

    2010-01-01

    Aldosterone is currently thought to exert its physiologic effects by activating epithelial mineralocorticoid receptors, and its pathologic effects on the cardiovascular system via mineralocorticoid receptors in the heart and blood vessels. Recent studies have extended this understanding to include a reevaluation of the roles of aldosterone and mineralocorticoid receptor activation in blood pressure control; the rapid, nongenomic effects of aldosterone; the role of cortisol as a mineralocorticoid receptor agonist under conditions of redox change/tissue damage/reactive oxygen species generation; the growing consensus that primary aldosteronism accounts for approximately 10% of all essential hypertension; recent new insights into the cardioprotective role of spironolactone; and the development of third- and fourth-generation mineralocorticoid receptor antagonists for use in cardiovascular and other inflammatory disease. These findings on aldosterone action and mineralocorticoid receptor blockade are analyzed in the context of the prevention and treatment of cardiovascular disease.

  17. Effects of ACTH on the last step of aldosterone biosynthesis.

    PubMed

    Cozza, E N; Ceballos, N R; Lantos, C P

    1989-12-01

    The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.

  18. Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells*S⃞

    PubMed Central

    Maron, Bradley A.; Zhang, Ying-Yi; Handy, Diane E.; Beuve, Annie; Tang, Shiow-Shih; Loscalzo, Joseph; Leopold, Jane A.

    2009-01-01

    Hyperaldosteronism is associated with impaired endothelium-dependent vascular reactivity owing to increased reactive oxygen species and decreased bioavailable nitric oxide (NO·); however, the effects of aldosterone on vasodilatory signaling pathways in vascular smooth muscle cells (VSMC) remain unknown. Soluble guanylyl cyclase (GC) is a heterodimer that is activated by NO· to convert cytosolic GTP to cGMP, a second messenger required for normal VSMC relaxation. Here, we show that aldosterone (10-9-10-7 mol/liter) diminishes GC activity by activating NADPH oxidase in bovine aortic VSMC to increase reactive oxygen species levels and induce oxidative posttranslational modification(s) of Cys-122, a β1-subunit cysteinyl residue demonstrated previously to modulate NO· sensing by GC. In VSMC treated with aldosterone, Western immunoblotting detected evidence of GC β1-subunit disulfide bonding, whereas mass spectrometry analysis of a homologous peptide containing the Cys-122-bearing sequence exposed to conditions of increased oxidant stress confirmed cysteinyl sulfinic acid (m/z 435), sulfonic acid (m/z 443), and disulfide (m/z 836) bond formation. The functional effect of these modifications was examined by transfecting COS-7 cells with wild-type GC or mutant GC containing an alanine substitution at Cys-122 (C122A). Exposure to aldosterone or hydrogen peroxide (H2O2) significantly decreased cGMP levels in cells expressing wild-type GC. In contrast, aldosterone or H2O2 did not influence cGMP levels in cells expressing the mutant C122A GC, confirming that oxidative modification of Cys-122 specifically impairs GC activity. These findings demonstrate that pathophysiologically relevant concentrations of aldosterone increase oxidant stress to convert GC to an NO·-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in VSMC. PMID:19141618

  19. Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation.

    PubMed

    Chiang, Chun-Te; Way, Tzong-Der; Tsai, Shang-Jie; Lin, Jen-Kun

    2007-12-22

    Fatty acid synthase (FAS) expression is markedly elevated in HER2-overexpressing breast cancer cells. In this study, diosgenin, a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin inhibited the phosphorylation of Akt and mTOR, and enhanced phosphorylation of JNK. The use of pharmacological inhibitors revealed that the modulation of Akt, mTOR and JNK phosphorylation was required for diosgenin-induced FAS suppression. Finally, we showed that diosgenin could enhance paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results suggested that diosgenin has the potential to advance as chemopreventive or chemotherapeutic agent for cancers that overexpress HER2.

  20. Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants.

    PubMed

    Sanmiya, K; Iwasaki, T; Matsuoka, M; Miyao, M; Yamamoto, N

    1997-02-28

    A cDNA encoding farnesyl diphosphate synthase (FPPS), a key enzyme in isoprenoid biosynthesis, was isolated from a cDNA library constructed from mRNA that had been prepared from etiolated rice (Oriza sativa L. variety Nipponbare) seedlings after three hours of illumination by a subtraction method. The putative polypeptide deduced from the 1289 bp nucleotide sequence consisted of 353 amino acids and had a molecular mass of 40 676 Da. The predicted amino acid sequence exhibited high homology to those of FPPS from Arabidopsis (73% to type 1, 72% to type 2) and white lupin (74%). Southern blot analysis showed that the rice genome might contain only one gene for FPPS. The highest level of expression of the gene was demonstrated in leaves by RNA blot analysis. Moreover, light, in particular blue light, effectively enhanced expression of the gene.

  1. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  2. Interacting influence of potassium and polychlorinated biphenyl on cortisol and aldosterone biosynthesis

    SciTech Connect

    Li, L.-A. . E-mail: lihann@nhri.org.tw; Lin, Tsu-Chun Emma

    2007-05-01

    Giving human adrenocortical H295R cells 14 mM KCl for 24 h significantly induced not only aldosterone biosynthesis but also cortisol biosynthesis. Pre-treating the cells with polychlorinated biphenyl 126 (PCB126) further increased potassium-induced aldosterone and cortisol productions in a dose-dependent manner, but all examined concentrations of PCB126 had little effect on the yields of precursor steroids progesterone and 17-OH-progesterone. Subsequent examinations revealed that CYP11B1 and CYP11B2 genes, responsible for the respective final steps of the cortisol and aldosterone biosynthetic pathways, exhibited increased responsiveness to PCB126 under high potassium. While 10{sup -5} M PCB126 was needed to induce a significant increase in the basal mRNA abundance of either gene, PCB126 could enhance potassium-induced mRNA expression of CYP11B1 at 10{sup -7} M and CYP11B2 at 10{sup -9} M. Actually, potassium and PCB126 synergistically upregulated mRNA expression of both genes. Potassium raised the transcriptional rates of CYP11B1 and CYP11B2 probably through a conserved Ad5 cis-element, whereas PCB126 appeared to regulate these two genes at the post-transcriptional level. Positive potassium-PCB126 synergism was also detected in CYP11B2 enzyme activity estimated by aldosterone/progesterone ratio. In contrast, potassium and PCB126 increased CYP11B1 enzyme activity or cortisol/17-OH-progesterone ratio additively. Moreover, potassium improved the time effect of PCB126 on gene expression and enzyme activity of CYP11B2, but not the PCB126 time response of CYP11B1. These data demonstrated that potassium differentially enhanced the potency of PCB126 to induce CYP11B1- and CYP11B2-mediated steroidogenesis.

  3. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner*

    PubMed Central

    Tirodkar, Tejas S.; Lu, Ping; Bai, Aiping; Scheffel, Matthew J.; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-01-01

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14–26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3–5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  4. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

  5. Hypercalcemia stimulates expression of intrarenal phospholipase A2 an