Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

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Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R. (Sanofi Aventis); (UMASS, Amherst)

2016-10-26

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

Roles of acid sphingomyelinase activation in neuronal cells apoptosis induced by microwave irradiation

International Nuclear Information System (INIS)

Zhang Lei; Xu Shangcheng; Zhang Guangbin; Yu Zhengping

2009-01-01

The present study is to examine the effect of microwave on acid sphingomyelinase (ASM) activity and expression, and to explore the role of ASM activation in neuronal cells apoptosis induced by microwave irradiation. Primary cultured hippocampal neurons were irradiated by 30 W/cm 2 microwave for 10 min, and ASM activity assay was used to investigate ASM activity alteration. RT-PCR and western blot were used to detect ASM mRNA and protein expression respectively. Apoptosis was observed by Hoechst 33342 fluorescence staining. ASM specific inhibitor imipramine was applied to inhibit ASM activation. It has been found that apoptosis rate of primary cultured hippocampal neurons increased significantly after microwave irradiation. ASM was activated while ASM mRNA and protein expression were upregulated in neurons after microwave irradiation. Pretreatment with imipramine could reverse neuronal apoptosis induced by microwave irradiation. Results show that microwave irradiation causes increment of ASM activation and expression and ASM activation is involved in microwave induced neuronal apoptosis. (authors)

Endothelial NLRP3 inflammasome activation and arterial neointima formation associated with acid sphingomyelinase during hypercholesterolemia

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Saisudha Koka

2017-10-01

Full Text Available The NLRP3 inflammasome has been reported to be activated by atherogenic factors, whereby endothelial injury and consequent atherosclerotic lesions are triggered in the arterial wall. However, the mechanisms activating and regulating NLRP3 inflammasomes remain poorly understood. The present study tested whether acid sphingomyelinase (ASM and ceramide associated membrane raft (MR signaling platforms contribute to the activation of NLRP3 inflammasomes and atherosclerotic lesions during hypercholesterolemia. We found that 7-ketocholesterol (7-Keto or cholesterol crystal (ChC markedly increased the formation and activation of NLRP3 inflammasomes in mouse carotid arterial endothelial cells (CAECs, as shown by increased colocalization of NLRP3 with ASC or caspase-1, enhanced caspase-1 activity and elevated IL-1β levels, which were markedly attenuated by mouse Asm siRNA, ASM inhibitor- amitriptyline, and deletion of mouse Asm gene. In CAECs with NLRP3 inflammasome formation, membrane raft (MR clustering with NADPH oxidase subunits was found remarkably increased as shown by CTXB (MR marker and gp91phox aggregation indicating the formation of MR redox signaling platforms. This MR clustering was blocked by MR disruptor (MCD, ROS scavenger (Tempol and TXNIP inhibitor (verapamil, accompanied by attenuation of 7-Keto or ChC-induced increase in caspase-1 activity. In animal experiments, Western diet fed mice with partially ligated left carotid artery (PLCA were found to have significantly increased neointimal formation, which was associated with increased NLRP3 inflammasome formation and IL-1β production in the intima of Asm+/+ mice but not in Asm-/- mice. These results suggest that Asm gene and ceramide associated MR clustering are essential to endothelial inflammasome activation and dysfunction in the carotid arteries, ultimately determining the extent of atherosclerotic lesions.

Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease.

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Lee, Jong Kil; Jin, Hee Kyung; Park, Min Hee; Kim, Bo-ra; Lee, Phil Hyu; Nakauchi, Hiromitsu; Carter, Janet E; He, Xingxuan; Schuchman, Edward H; Bae, Jae-sung

2014-07-28

In Alzheimer's disease (AD), abnormal sphingolipid metabolism has been reported, although the pathogenic consequences of these changes have not been fully characterized. We show that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, leading to defective autophagic degradation due to lysosomal depletion. Partial genetic inhibition of ASM (ASM(+/-)) in a mouse model of familial AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autophagocytic defect by restoring lysosomal biogenesis, resulting in improved AD clinical and pathological findings, including reduction of amyloid-β (Aβ) deposition and improvement of memory impairment. Similar effects were noted after pharmacologic restoration of ASM to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition. Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM inhibition is a potential new therapeutic intervention for the disease. © 2014 Lee et al.

Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

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Ranganath, Prajnya; Matta, Divya; Bhavani, Gandham SriLakshmi; Wangnekar, Savita; Jain, Jamal Mohammed Nurul; Verma, Ishwar C; Kabra, Madhulika; Puri, Ratna Dua; Danda, Sumita; Gupta, Neerja; Girisha, Katta M; Sankar, Vaikom H; Patil, Siddaramappa J; Ramadevi, Akella Radha; Bhat, Meenakshi; Gowrishankar, Kalpana; Mandal, Kausik; Aggarwal, Shagun; Tamhankar, Parag Mohan; Tilak, Preetha; Phadke, Shubha R; Dalal, Ashwin

2016-10-01

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis

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Taniguchi, M; Ogiso, H; Takeuchi, T; Kitatani, K; Umehara, H; Okazaki, T

2015-01-01

We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM?ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis pro...

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

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Oninla, Vincent O; Breiden, Bernadette; Babalola, Jonathan O; Sandhoff, Konrad

2014-12-01

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

The Common Acid Sphingomyelinase Polymorphism p.G508R is Associated with Self-Reported Allergy

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Martin Reichel

2014-06-01

Full Text Available Background: Acid sphingomyelinase (ASM is a key regulator of ceramide-dependent signalling pathways. Among others, activation of ASM can be induced by CD95 or cytokine signalling and by cellular stress resulting from inflammation or infection. Increased ASM activity was observed in a variety of human diseases including inflammatory and neuropsychiatric disorders. We hypothesized that basal ASM activity might influence the susceptibility for common human diseases. Methods: The general health condition of 100 young people was assessed using a questionnaire. The ASM polymorphism rs1050239 (c.1522G>A; encoding p.G508R was determined from genomic DNA. Activities of secretory (S- and lysosomal (L- ASM were measured in blood plasma and peripheral blood cells respectively. Results: The polymorphism rs1050239 was significantly associated with self-reported allergy (p=4.68×10-4; adjusted p-value for multiple testing 0.007. Allergy was more prevalent in carriers of the minor A allele compared to non-carriers (p=0.00015; odds ratio=6.5, 95% CI 2.15-21.7. S-ASM activity was significantly associated with rs1050239 (p=5.3×10-7 and decreased with the number of A alleles in a gene-dosage dependent manner. In allergic patients, S-ASM activity was moderately decreased (p=0.034. L-ASM activity was significantly lower in subjects homozygous for the minor A allele (p=0.025 but not different between allergic and non-allergic subjects (p=0.318. Conclusion: Our analysis provides evidence for an involvement of ASM in the pathophysiology of allergy, which is in line with previous reports addressing the role of sphingolipids in this disorder. Further studies should clarify the mechanism linking rs1050239 to allergy. The ASM pathway might be useful for predicting allergic disposition and disease course and as a therapeutic target.

Comparative analysis of acid sphingomyelinase distribution in the CNS of rats and mice following intracerebroventricular delivery.

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Christopher M Treleaven

Full Text Available Niemann-Pick A (NPA disease is a lysosomal storage disorder (LSD caused by a deficiency in acid sphingomyelinase (ASM activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO mice could be partially alleviated by intracerebroventricular (ICV infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat.

Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals.

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Edmead, C E; Patel, Y I; Wilson, A; Boulougouris, G; Hall, N D; Ward, S G; Sansom, D M

1996-10-15

A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways.

Delivery of acid sphingomyelinase in normal and niemann-pick disease mice using intercellular adhesion molecule-1-targeted polymer nanocarriers.

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Garnacho, Carmen; Dhami, Rajwinder; Simone, Eric; Dziubla, Thomas; Leferovich, John; Schuchman, Edward H; Muzykantov, Vladimir; Muro, Silvia

2008-05-01

Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

Elevation of Serum Acid Sphingomyelinase Activity in Children with Acute Respiratory Syncytial Virus Bronchiolitis.

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Yoshida, Shuichiro; Noguchi, Atsuko; Kikuchi, Wataru; Fukaya, Hiroshi; Igarashi, Kiyoshi; Takahashi, Tsutomu

2017-12-01

Acid sphingomyelinase (ASM) is a lysosomal enzyme that hydrolyzes sphingomyelin into ceramide, a bioactive lipid to regulate cellular physiological functions. Thus, ASM activation has been reported as a key event in pathophysiological reactions including inflammation, cytokine release, oxidative stress, and endothelial damage in human diseases. Since ASM activation is associated with extracellular ASM secretion through unknown mechanisms, it can be detected by recognizing the elevation of secretory ASM (S-ASM) activity. Serum S-ASM activity has been reported to increase in chronic diseases, acute cardiac diseases, and systemic inflammatory diseases. However, the serum S-ASM has not been investigated in common acute illness. This study was designed to evaluate serum S-ASM activity in children with common acute illness. Fifty children with common acute illness and five healthy children were included in this study. The patients were categorized into five groups based on clinical diagnoses: acute respiratory syncytial virus (RSV) bronchiolitis, adenovirus infection, streptococcal infection, asthma, and other infections due to unknown origin. The serum S-ASM activity was significantly elevated at 6.9 ± 1.6 nmol/0.1 mL/6 h in the group of acute RSV bronchiolitis patients compared with healthy children who had a mean level of 1.8 ± 0.8 nmol/0.1 mL/6 h (p ASM activity was not significantly elevated. The results suggest an association of ASM activation with RSV infection, a cause for common acute illness. This is the first report to describe the elevation of serum S-ASM activity in respiratory tract infection.

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

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Zhou, Yuetao; Salker, Madhuri S; Walker, Britta; Münzer, Patrick; Borst, Oliver; Gawaz, Meinrad; Gulbins, Erich; Singh, Yogesh; Lang, Florian

2016-01-01

Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-β and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1β, IL-6 and IL-17 but not IFN-γ mRNA levels. ASM is a negative regulator of natural and iTreg. © 2016 The Author(s) Published by S. Karger AG, Basel.

Seven novel mutations of the SMPD1 gene in four Chinese patients with Niemann-Pick disease type A and prenatal diagnosis for four fetuses.

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Ding, Yuan; Li, Xiyuan; Liu, Yupeng; Hua, Ying; Song, Jinqing; Wang, Liwen; Li, Mengqiu; Qin, Yaping; Yang, Yanling

2016-04-01

Niemann-Pick disease type A (NPD-A) is a rare autosomal recessive lysosomal storage disorder caused by acid sphingomyelinase deficiency. Only a few cases have been documented in mainland China, and prenatal diagnosis has not been performed to date. In this study, the clinical and laboratory features of four Chinese patients with early-onset NPD-A were summarized. Four patients with NPD-A were the firstborns of non-consanguineous parents from four unrelated Chinese families. Bone marrow analysis, acid sphingomyelinase assay and genetic studies were performed. SMPD1 gene studies on amniocytes were performed for the prenatal diagnosis of four fetuses from three families. Four patients were admitted at the age of 1-10 months due to jaundice, hepatosplenomegaly and psychomotor retardation. Liver histopathological analysis revealed glucolipid accumulation. Massive foamy histiocytes were found in the bone marrow. Acid sphingomyelinase activities of peripheral blood leukocytes were significantly decreased (4.05-21.9 nmol/h/mg protein, normal range 216.1-950.9 nmol/h/mg protein). Seven novel mutations (c.518-519insT, c.562_563insC, c.792Gdel, c.949G>A, c.1487_1499delACCGTGTGTACCA, c.1495T>C and c.1670T>C) of the SMPD1 gene were identified in four patients. Only one fetus had two mutations of the SMPD1 gene of amniocytes. The results suggested that the fetus was affected by NPD-A. The mother chose artificial abortion. The other three fetuses were not affected by NPD-A. No mutation of the SMPD1 gene was detected in the cultured amniocytes from the mothers. Postnatal genetic analysis and normal development of the three infants confirmed the prenatal diagnosis. Seven novel mutations associated with NPD-A were identified in the Chinese population. Prenatal diagnosis for four fetuses of three families was successfully performed by amniocyte gene analysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Acid sphingomyelinase (Asm) deficiency patients in The Netherlands and Belgium: disease spectrum and natural course in attenuated patients.

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Hollak, C E M; de Sonnaville, E S V; Cassiman, D; Linthorst, G E; Groener, J E; Morava, E; Wevers, R A; Mannens, M; Aerts, J M F G; Meersseman, W; Akkerman, E; Niezen-Koning, K E; Mulder, M F; Visser, G; Wijburg, F A; Lefeber, D; Poorthuis, B J H M

2012-11-01

Niemann-Pick disease (NPD) is a neurovisceral lysosomal storage disorder caused by acid sphingomyelinase (ASM) deficiency, which can be categorized as either Niemann-Pick disease type A [NPD-A], with progressive neurological disease and death in early childhood, or as Niemann-Pick disease type B [NPD-B], with a more variable spectrum of manifestations. Enzyme replacement therapy (ERT) with recombinant sphingomyelinase is currently studied as potential treatment for NPD-B patients. The objective of this study is to characterize the clinical features of patients with ASM deficiency in the Netherlands and Belgium with focus on the natural disease course of NPD-B patients. Prospective and retrospective data on ASM deficient patients were collected in The Netherlands and part of Belgium. Patients with NPD-B that could be followed prospectively were evaluated every 6-12 months for pulmonary function tests, 6 minute walk test (6 MWT), imaging (bone marrow infiltration measured by QCSI, organ volumes by MRI and CT scan of the lungs) and biochemical markers. Twenty-five patients with ASM deficiency were identified (13 males, 12 females, median age 13years, range 1-59 years). Nine patients had died at the time of the study, including four NPD-A patients at the age of 1,1, 2, 3 and five NPDB patents at the age of 5, 6, 43, 56 and 60 years. There was a high prevalence of homozygosity and compound heterozygosity for the common p.Arg608del mutation in 43% and 19% of NPD-B patients, respectively. In NPD-B patients, thrombocytopenia was present in most, while anemia and leucopenia were less common (33% and 6 % respectively). HDL cholesterol was reduced in most patients. Pulmonary disease was severe in several patients. Follow-up up to 11 years revealed a gradual decrease in platelet count. Detailed investigations in 6 NPD-B patients with follow-up in 4 patients revealed remarkable stable disease parameters up to 6 years, with some decline in pulmonary function and 6 MWT. Bone

Alpha-mangostin attenuates diabetic nephropathy in association with suppression of acid sphingomyelianse and endoplasmic reticulum stress.

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Liu, Tingting; Duan, Wang; Nizigiyimana, Paul; Gao, Lin; Liao, Zhouning; Xu, Boya; Liu, Lerong; Lei, Minxiang

2018-02-05

Diabetic nephropathy is a common complication of diabetes, but there are currently few treatment options. The aim of this study was to gain insight into the effect of alpha-mangostin on diabetic nephropathy and possible related mechanisms. Goto-Kakizaki rats were used as a diabetic model and received alpha-mangostin or desipramine treatment with normal saline as a control. Ten age-matched Sprague Dawley rats were used as normal controls and treated with normal saline. At week 12, blood glucose, albuminuria, apoptosis and renal pathologic changes were assessed. Protein levels for acid sphingomyelinase, glucose-regulated protein 78, phosphorylated PKR-like ER-resident kinase, activated transcription factor 4, CCAAT/enhancer-binding protein, homologous protein), and cleaved-caspase12 were measured. The level of acid sphingomyelinase was significantly increased, and ER stress was activated in diabetic rat kidneys when compared to the control animals. When acid sphingomyelinase was inhibited by alpha-mangostin, the expression of ER stress-related proteins was down-regulated in association with decreased levels of diabetic kidney injury. Alpha-mangostin, an acid sphingomyelinase inhibitor plays a protective role in diabetic neuropathy by relieving ER stress induced-renal cell apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

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Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

2004-11-15

Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

Structural basis of the phospholipase C activity in neutral sphingomyelinase from Bacillus cereus

International Nuclear Information System (INIS)

Ago, Hideo; Miyano, Masashi

2007-01-01

Degradation of cell membrane and mucosa, of which phospholipids are major components, and production of lipid mediators are roles of phospholipases from pathogenic bacteria to grow, survive and spread in the host organism. The studies on the enzymes the important for the pathobiology of bacterial infectious disease. The crystal structure of Sphingomyelinase from Bacillus cereus revealed the structure basis of the phospholipase C and hemolysis activities in a divalent cation dependent manner. The water-bridged double divalent cations were concluded to be the catalytic architecture to the phospholipase C activity. In addition, the β-hairpin structure with aromatic amino acid residues was shown to be involved in the membrane binding of the enzyme as a part of the hemolysis activity. (author)

Sphingomyelinase Activity of Trichomonas vaginalis Extract and Subfractions

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Francisco González-Salazar

2013-01-01

Full Text Available Trichomoniasis is one of the most common acute sexually transmitted curable diseases, and it is disseminated worldwide generating more than 170 million cases annually. Trichomonas vaginalis is the parasite that causes trichomoniasis and has the ability to destroy cell monolayers of the vaginal mucosa in vitro. Sphingomyelinases (SMase are enzymes that catalyze the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Ceramide appears to be a second messenger lipid in programmed apoptosis, cell differentiation, and cell proliferation. Sphingomyelinase is probably a major source of ceramide in cells. Signal transduction mediated by ceramide leads cells to produce cytokine induced apoptosis during several inflammatory responses. SMase are also relevant toxins in several microorganisms. The main objective of this research is to identify SMase activity of T. vaginalis in the total extract (TE, P30, and S30 subfractions from brooked trophozoites. It was found that these fractions of T. vaginalis have SMase activity, which comes principally from P30 subfraction and was mainly type C. Enzymatic activity of SMase increased linearly with time and is pH dependent with two peaks by pH 5.5 and pH 7.5. The addition of manganese to the reaction mixture increased the SMase activity by 1.97.

ASM-3 acid sphingomyelinase functions as a positive regulator of the DAF-2/AGE-1 signaling pathway and serves as a novel anti-aging target.

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Yongsoon Kim

Full Text Available In C. elegans, the highly conserved DAF-2/insulin/insulin-like growth factor 1 receptor signaling (IIS pathway regulates longevity, metabolism, reproduction and development. In mammals, acid sphingomyelinase (ASM is an enzyme that hydrolyzes sphingomyelin to produce ceramide. ASM has been implicated in CD95 death receptor signaling under certain stress conditions. However, the involvement of ASM in growth factor receptor signaling under physiological conditions is not known. Here, we report that in vivo ASM functions as a positive regulator of the DAF-2/IIS pathway in C. elegans. We have shown that inactivation of asm-3 extends animal lifespan and promotes dauer arrest, an alternative developmental process. A significant cooperative effect on lifespan is observed between asm-3 deficiency and loss-of-function alleles of the age-1/PI 3-kinase, with the asm-3; age-1 double mutant animals having a mean lifespan 259% greater than that of the wild-type animals. The lifespan extension phenotypes caused by the loss of asm-3 are dependent on the functions of daf-16/FOXO and daf-18/PTEN. We have demonstrated that inactivation of asm-3 causes nuclear translocation of DAF-16::GFP protein, up-regulates endogenous DAF-16 protein levels and activates the downstream targeting genes of DAF-16. Together, our findings reveal a novel role of asm-3 in regulation of lifespan and diapause by modulating IIS pathway. Importantly, we have found that two drugs known to inhibit mammalian ASM activities, desipramine and clomipramine, markedly extend the lifespan of wild-type animals, in a manner similar to that achieved by genetic inactivation of the asm genes. Our studies illustrate a novel strategy of anti-aging by targeting ASM, which may potentially be extended to mammals.

ASM-3 acid sphingomyelinase functions as a positive regulator of the DAF-2/AGE-1 signaling pathway and serves as a novel anti-aging target.

Science.gov (United States)

Kim, Yongsoon; Sun, Hong

2012-01-01

In C. elegans, the highly conserved DAF-2/insulin/insulin-like growth factor 1 receptor signaling (IIS) pathway regulates longevity, metabolism, reproduction and development. In mammals, acid sphingomyelinase (ASM) is an enzyme that hydrolyzes sphingomyelin to produce ceramide. ASM has been implicated in CD95 death receptor signaling under certain stress conditions. However, the involvement of ASM in growth factor receptor signaling under physiological conditions is not known. Here, we report that in vivo ASM functions as a positive regulator of the DAF-2/IIS pathway in C. elegans. We have shown that inactivation of asm-3 extends animal lifespan and promotes dauer arrest, an alternative developmental process. A significant cooperative effect on lifespan is observed between asm-3 deficiency and loss-of-function alleles of the age-1/PI 3-kinase, with the asm-3; age-1 double mutant animals having a mean lifespan 259% greater than that of the wild-type animals. The lifespan extension phenotypes caused by the loss of asm-3 are dependent on the functions of daf-16/FOXO and daf-18/PTEN. We have demonstrated that inactivation of asm-3 causes nuclear translocation of DAF-16::GFP protein, up-regulates endogenous DAF-16 protein levels and activates the downstream targeting genes of DAF-16. Together, our findings reveal a novel role of asm-3 in regulation of lifespan and diapause by modulating IIS pathway. Importantly, we have found that two drugs known to inhibit mammalian ASM activities, desipramine and clomipramine, markedly extend the lifespan of wild-type animals, in a manner similar to that achieved by genetic inactivation of the asm genes. Our studies illustrate a novel strategy of anti-aging by targeting ASM, which may potentially be extended to mammals.

ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target

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Kim, Yongsoon; Sun, Hong

2012-01-01

In C. elegans, the highly conserved DAF-2/insulin/insulin-like growth factor 1 receptor signaling (IIS) pathway regulates longevity, metabolism, reproduction and development. In mammals, acid sphingomyelinase (ASM) is an enzyme that hydrolyzes sphingomyelin to produce ceramide. ASM has been implicated in CD95 death receptor signaling under certain stress conditions. However, the involvement of ASM in growth factor receptor signaling under physiological conditions is not known. Here, we report that in vivo ASM functions as a positive regulator of the DAF-2/IIS pathway in C. elegans. We have shown that inactivation of asm-3 extends animal lifespan and promotes dauer arrest, an alternative developmental process. A significant cooperative effect on lifespan is observed between asm-3 deficiency and loss-of-function alleles of the age-1/PI 3-kinase, with the asm-3; age-1 double mutant animals having a mean lifespan 259% greater than that of the wild-type animals. The lifespan extension phenotypes caused by the loss of asm-3 are dependent on the functions of daf-16/FOXO and daf-18/PTEN. We have demonstrated that inactivation of asm-3 causes nuclear translocation of DAF-16::GFP protein, up-regulates endogenous DAF-16 protein levels and activates the downstream targeting genes of DAF-16. Together, our findings reveal a novel role of asm-3 in regulation of lifespan and diapause by modulating IIS pathway. Importantly, we have found that two drugs known to inhibit mammalian ASM activities, desipramine and clomipramine, markedly extend the lifespan of wild-type animals, in a manner similar to that achieved by genetic inactivation of the asm genes. Our studies illustrate a novel strategy of anti-aging by targeting ASM, which may potentially be extended to mammals. PMID:23049887

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

NARCIS (Netherlands)

Duan, R. D.; Verkade, H. J.; Cheng, Y.; Havinga, R.; Nilsson, A.

Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-CDase) in the intestinal microvillar membrane are responsible for dietary sphingomyelin digestion. The activities of the enzymes require the presence of bile salt, and the enzymes can be released into the gut lumen in active forms by

Structural and functional analysis of the ASM p.Ala359Asp mutant that causes acid sphingomyelinase deficiency.

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Acuña, Mariana; Castro-Fernández, Víctor; Latorre, Mauricio; Castro, Juan; Schuchman, Edward H; Guixé, Victoria; González, Mauricio; Zanlungo, Silvana

2016-10-21

Niemann-Pick disease (NPD) type A and B are recessive hereditary disorders caused by deficiency in acid sphingomyelinase (ASM). The p.Ala359Asp mutation has been described in several patients but its functional and structural effects in the protein are unknown. In order to characterize this mutation, we modeled the three-dimensional ASM structure using the recent available crystal of the mammalian ASM as a template. We found that the p.Ala359Asp mutation is localized in the hydrophobic core and far from the sphingomyelin binding site. However, energy function calculations using statistical potentials indicate that the mutation causes a decrease in ASM stability. Therefore, we investigated the functional effect of the p.Ala359Asp mutation in ASM expression, secretion, localization and activity in human fibroblasts. We found a 3.8% residual ASM activity compared to the wild-type enzyme, without changes in the other parameters evaluated. These results support the hypothesis that the p.Ala359Asp mutation causes structural alterations in the hydrophobic environment where ASM is located, decreasing its enzymatic activity. A similar effect was observed in other previously described NPDB mutations located outside the active site of the enzyme. This work shows the first full size ASM mutant model describe at date, providing a complete analysis of the structural and functional effects of the p.Ala359Asp mutation over the stability and activity of the enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.

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Kolesnick, R N; Clegg, S

1988-05-15

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C

Neutral Sphingomyelinase (SMPD3) Deficiency Causes a Novel Form of Chondrodysplasia and Dwarfism That Is Rescued by Col2A1-Driven smpd3 Transgene Expression

OpenAIRE

Stoffel, Wilhelm; Jenke, Britta; Holz, Barbara; Binczek, Erika; Günter, Robert Heinz; Knifka, Jutta; Koebke, Jürgen; Niehoff, Anja

2007-01-01

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, ass...

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

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Roberto P Stock

Full Text Available The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1 ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2 the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3 in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Extracellular Sphingomyelinase Rv0888 of Mycobacterium tuberculosis Contributes to Pathological Lung Injury of Mycobacterium smegmatis in Mice via Inducing Formation of Neutrophil Extracellular Traps.

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Dang, Guanghui; Cui, Yingying; Wang, Lei; Li, Tiantian; Cui, Ziyin; Song, Ningning; Chen, Liping; Pang, Hai; Liu, Siguo

2018-01-01

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which mainly causes pulmonary injury and tubercles. Although macrophages are generally considered to harbor the main cells of M. tuberculosis , new evidence suggests that neutrophils are rapidly recruited to the infected lung. M. tuberculosis itself, or its early secreted antigenic target protein 6 (ESAT-6), can induce formation of neutrophil extracellular traps (NETs). However, NETs trap mycobacteria but are unable to kill them. The role of NETs' formation in the pathogenesis of mycobacteria remains unclear. Here, we report a new M. tuberculosis extracellular factor, bifunctional enzyme Rv0888, with both nuclease and sphingomyelinase activities. Rv0888 sphingomyelinase activity can induce NETs' formation in vitro and in the lung of the mice and enhance the colonization ability of Mycobacterium smegmatis in the lungs of mice. Mice infected by M. smegmatis harboring Rv0888 sphingomyelinase induced pathological injury and inflammation of the lung, which was mainly mediated by NETs, induced by Rv0888 sphingomyelinase, associated protein (myeloperoxidase) triggered caspase-3. In summary, the study sheds new light on the pathogenesis of mycobacteria and reveals a novel target for TB treatment.

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate

DEFF Research Database (Denmark)

Stock, Roberto; Brewer, Jonathan R.; Wagner, Kerstin

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model...... membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy...... and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing...

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development

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Stoffel, Wilhelm; Jenke, Britta; Blöck, Barbara; Zumbansen, Markus; Koebke, Jürgen

2005-01-01

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3–/– and smpd2–/–smpd3–/– double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3–/– mouse might mimic a form of human combined pituitary hormone deficiency. PMID:15764706

Acid sphingomyelinase activity as an indicator of the cell stress in HPV-positive and HPV-negative head and neck squamous cell carcinoma.

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Gerle, Mirko; Medina, Tuula Peñate; Gülses, Aydin; Chu, Hanwen; Naujokat, Hendrik; Wiltfang, Jörg; Açil, Yahya

2018-03-21

Human papillomavirus (HPV) infection, especially HPV-16 and HPV-18, has been increasingly associated with head and neck squamous cell carcinoma. The treatment of HPV-positive squamous cell carcinoma has a better response to both radiotherapy and chemotherapy and presents a better prognosis for the patient. Defining the underlying mechanism of the difference might help in developing future treatment options and could be an important factor in personal therapy planning. Endogenously secreted acid sphingomyelinase (ASMase) levels in the cellular stress caused by irradiation and cisplatin were investigated. MTT assay was performed to evaluate the viability of the treated cells. Keratinocytes were used to evaluate the effects of radiation on normal tissues. Irradiation caused a dose-dependent increase in ASMase activity in both SCC9 HPV-negative, and UDSCC2 HPV-positive cells. ASMase activity in UDSCC2 cells was significantly higher than that in SCC9 cells. UDSCC cells were more sensitive to cisplatin treatment than SCC cells, and the dose-response in the activity was observed in long-time treatments when high doses of cisplatin were used. The results of the current study have clearly showed that HPV positivity should be considered as one of the determinative factors which should be considered when tumor treatments are planned. However, further studies are needed to determine the differences in cellular responses and pathways among HPV-negative and HPV-positive cells.

Concerted action of sphingomyelinase and non-hemolytic enterotoxin in pathogenic Bacillus cereus.

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Viktoria M Doll

Full Text Available Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe, which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

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Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest.

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Shamseddine, A A; Clarke, C J; Carroll, B; Airola, M V; Mohammed, S; Rella, A; Obeid, L M; Hannun, Y A

2015-10-29

Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.

Molecular modeling of human neutral sphingomyelinase provides insight into its molecular interactions.

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Dinesh; Goswami, Angshumala; Suresh, Panneer Selvam; Thirunavukkarasu, Chinnasamy; Weiergräber, Oliver H; Kumar, Muthuvel Suresh

2011-01-01

The neutral sphingomyelinase (N-SMase) is considered a major candidate for mediating the stress-induced production of ceramide, and it plays an important role in cell-cycle arrest, apoptosis, inflammation, and eukaryotic stress responses. Recent studies have identified a small region at the very N-terminus of the 55 kDa tumour necrosis factor receptor (TNF-R55), designated the neutral sphingomyelinase activating domain (NSD) that is responsible for the TNF-induced activation of N-SMase. There is no direct association between TNF-R55 NSD and N-SMase; instead, a protein named factor associated with N-SMase activation (FAN) has been reported to couple the TNF-R55 NSD to N-SMase. Since the three-dimensional fold of N-SMase is still unknown, we have modeled the structure using the protein fold recognition and threading method. Moreover, we propose models for the TNF-R55 NSD as well as the FAN protein in order to study the structural basis of N-SMase activation and regulation. Protein-protein interaction studies suggest that FAN is crucially involved in mediating TNF-induced activation of the N-SMase pathway, which in turn regulates mitogenic and proinflammatory responses. Inhibition of N-SMase may lead to reduction of ceramide levels and hence may provide a novel therapeutic strategy for inflammation and autoimmune diseases. Molecular dynamics (MD) simulations were performed to check the stability of the predicted model and protein-protein complex; indeed, stable RMS deviations were obtained throughout the simulation. Furthermore, in silico docking of low molecular mass ligands into the active site of N-SMase suggests that His135, Glu48, Asp177, and Asn179 residues play crucial roles in this interaction. Based on our results, these ligands are proposed to be potent and selective N-SMase inhibitors, which may ultimately prove useful as lead compounds for drug development.

Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis.

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Taniguchi, M; Ogiso, H; Takeuchi, T; Kitatani, K; Umehara, H; Okazaki, T

2015-04-09

We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM-ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM-ceramide-CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.

c-Abl is an upstream regulator of acid sphingomyelinase in apoptosis induced by inhibition of integrins αvβ3 and αvβ5.

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Xiuhai Ren

Full Text Available Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM, and increased ceramides C(16, C(18:0, C(24:0 and C(24:1 while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.

Four Novel p.N385K, p.V36A, c.1033–1034insT and c.1417–1418delCT Mutations in the Sphingomyelin Phosphodiesterase 1 (SMPD1 Gene in Patients with Types A and B Niemann-Pick Disease (NPD

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Masoumeh Dehghan Manshadi

2015-03-01

Full Text Available Background: Types A and B Niemann-Pick disease (NPD are autosomal-recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1 gene. Methods: In order to determine the prevalence and distribution of SMPD1 gene mutations, the genomic DNA of 15 unrelated Iranian patients with types A and B NPD was examined using PCR, DNA sequencing and bioinformatics analysis. Results: Of 8 patients with the p.G508R mutation, 5 patients were homozygous, while the other 3 were heterozygous. One patient was heterozygous for both the p.N385K and p.G508R mutations. Another patient was heterozygous for both the p.A487V and p.G508R mutations. Two patients (one homozygous and one heterozygous showed the p.V36A mutation. One patient was homozygous for the c.1033–1034insT mutation. One patient was homozygous for the c.573delT mutation, and 1 patient was homozygous for the c.1417–1418delCT mutation. Additionally, bioinformatics analysis indicated that two new p.V36A and p.N385K mutations decreased the acid sphingomyelinase (ASM protein stability, which might be evidence to suggest the pathogenicity of these mutations. Conclusion: with detection of these new mutations, the genotypic spectrum of types A and B NPD is extended, facilitating the definition of disease-related mutations. However, more research is essential to confirm the pathogenic effect of these mutations.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

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Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

2016-07-01

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Uncovering co-expression gene network modules regulating fruit acidity in diverse apples.

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Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Zhong, Gan-Yuan; Xu, Kenong

2015-08-16

Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background.

DNA methylation of amino acid transporter genes in the human placenta.

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Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

2017-12-01

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

Nitric Oxide Generated by Tumor-Associated Macrophages Is Responsible for Cancer Resistance to Cisplatin and Correlated With Syntaxin 4 and Acid Sphingomyelinase Inhibition

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Cristiana Perrotta

2018-05-01

Full Text Available Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO-generating enzyme inducible nitric oxide synthase (iNOS. NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP. Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase, which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG, inducing phosphorylation of syntaxin 4 (synt4, a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically

The Activity of the Neutral Sphingomyelinase Is Important in T Cell Recruitment and Directional Migration

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Lena Collenburg

2017-08-01

Full Text Available Breakdown of sphingomyelin as catalyzed by the activity of sphingomyelinases profoundly affects biophysical properties of cellular membranes which is particularly important with regard to compartmentalization of surface receptors and their signaling relay. As it is activated both upon TCR ligation and co-stimulation in a spatiotemporally controlled manner, the neutral sphingomyelinase (NSM has proven to be important in T cell activation, where it appears to play a particularly important role in cytoskeletal reorganization and cell polarization. Because these are important parameters in directional T cell migration and motility in tissues, we analyzed the role of the NSM in these processes. Pharmacological inhibition of NSM interfered with early lymph node homing of T cells in vivo indicating that the enzyme impacts on endothelial adhesion, transendothelial migration, sensing of chemokine gradients or, at a cellular level, acquisition of a polarized phenotype. NSM inhibition reduced adhesion of T cells to TNF-α/IFN-γ activated, but not resting endothelial cells, most likely via inhibiting high-affinity LFA-1 clustering. NSM activity proved to be highly important in directional T cell motility in response to SDF1-α, indicating that their ability to sense and translate chemokine gradients might be NSM dependent. In fact, pharmacological or genetic NSM ablation interfered with T cell polarization both at an overall morphological level and redistribution of CXCR4 and pERM proteins on endothelial cells or fibronectin, as well as with F-actin polymerization in response to SDF1-α stimulation, indicating that efficient directional perception and signaling relay depend on NSM activity. Altogether, these data support a central role of the NSM in T cell recruitment and migration both under homeostatic and inflamed conditions by regulating polarized redistribution of receptors and their coupling to the cytoskeleton.

Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil.

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Pham, Anh-Tung; Shannon, J Grover; Bilyeu, Kristin D

2012-08-01

High oleic acid soybeans were produced by combining mutant FAD2-1A and FAD2-1B genes. Despite having a high oleic acid content, the linolenic acid content of these soybeans was in the range of 4-6 %, which may be high enough to cause oxidative instability of the oil. Therefore, a study was conducted to incorporate one or two mutant FAD3 genes into the high oleic acid background to further reduce the linolenic acid content. As a result, soybean lines with high oleic acid and low linolenic acid (HOLL) content were produced using different sources of mutant FAD2-1A genes. While oleic acid content of these HOLL lines was stable across two testing environments, the reduction of linolenic acid content varied depending on the number of mutant FAD3 genes combined with mutant FAD2-1 genes, on the severity of mutation in the FAD2-1A gene, and on the testing environment. Combination of two mutant FAD2-1 genes and one mutant FAD3 gene resulted in less than 2 % linolenic acid content in Portageville, Missouri (MO) while four mutant genes were needed to achieve the same linolenic acid in Columbia, MO. This study generated non-transgenic soybeans with the highest oleic acid content and lowest linolenic acid content reported to date, offering a unique alternative to produce a fatty acid profile similar to olive oil.

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

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Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

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Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Gene coexpression network analysis of fruit transcriptomes uncovers a possible mechanistically distinct class of sugar/acid ratio-associated genes in sweet orange.

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Qiao, Liang; Cao, Minghao; Zheng, Jian; Zhao, Yihong; Zheng, Zhi-Liang

2017-10-30

The ratio of sugars to organic acids, two of the major metabolites in fleshy fruits, has been considered the most important contributor to fruit sweetness. Although accumulation of sugars and acids have been extensively studied, whether plants evolve a mechanism to maintain, sense or respond to the fruit sugar/acid ratio remains a mystery. In a prior study, we used an integrated systems biology tool to identify a group of 39 acid-associated genes from the fruit transcriptomes in four sweet orange varieties (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). We reanalyzed the prior sweet orange fruit transcriptome data, leading to the identification of 72 genes highly correlated with the fruit sugar/acid ratio. The majority of these sugar/acid ratio-related genes are predicted to be involved in regulatory functions such as transport, signaling and transcription or encode enzymes involved in metabolism. Surprisingly, only three of these sugar/acid ratio-correlated genes are weakly correlated with sugar level and none of them overlaps with the acid-associated genes. Weighted Gene Coexpression Network Analysis (WGCNA) has revealed that these genes belong to four modules, Blue, Grey, Brown and Turquoise, with the former two modules being unique to the sugar/acid ratio control. Our results indicate that orange fruits contain a possible mechanistically distinct class of genes that may potentially be involved in maintaining fruit sugar/acid ratios and/or responding to the cellular sugar/acid ratio status. Therefore, our analysis of orange transcriptomes provides an intriguing insight into the potentially novel genetic or molecular mechanisms controlling the sugar/acid ratio in fruits.

[Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

Science.gov (United States)

Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

2014-09-01

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

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

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

2014-11-01

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

Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

Science.gov (United States)

Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

2015-12-01

In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

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Mahdi Ebrahimi

2014-09-01

Full Text Available Alteration of the lipid content and fatty acid (FA composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA and α-linolenic acid (LNA for 100 days. Inclusion of flaxseed oil increased (p < 0.05 the α-linolenic acid (C18:3n-3 concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05 decreased the arachidonic acid (C20:4n-6 and conjugated linolenic acid (CLA c-9 t-11 content in the ST muscle. There was a significant (p < 0.05 upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression.

Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio: evidence for subfunctionalization or neofunctionalization of duplicated genes

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Denovan-Wright Eileen M

2009-09-01

Full Text Available Abstract Background In the Duplication-Degeneration-Complementation (DDC model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps genes by dietary fatty acids (FAs in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid, sunflower oil (12% lipid, rich in linoleic acid, linseed oil (12% lipid, rich in linolenic acid, or low fat (4% lipid, low fat diet for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. Result FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR. In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases

Isolation of a novel abscisic acid stress ripening ( OsASR ) gene ...

African Journals Online (AJOL)

Isolation of a novel abscisic acid stress ripening ( OsASR ) gene from rice and analysis of the response of this gene to abiotic stresses. ... The cDNA with the whole open reading frame (ORF) was amplified by PCR and cloned. Sequence analysis showed that the cDNA encodes a protein of 284 amino acid residues with ...

Coordinations between gene modules control the operation of plant amino acid metabolic networks

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Galili Gad

2009-01-01

Full Text Available Abstract Background Being sessile organisms, plants should adjust their metabolism to dynamic changes in their environment. Such adjustments need particular coordination in branched metabolic networks in which a given metabolite can be converted into multiple other metabolites via different enzymatic chains. In the present report, we developed a novel "Gene Coordination" bioinformatics approach and use it to elucidate adjustable transcriptional interactions of two branched amino acid metabolic networks in plants in response to environmental stresses, using publicly available microarray results. Results Using our "Gene Coordination" approach, we have identified in Arabidopsis plants two oppositely regulated groups of "highly coordinated" genes within the branched Asp-family network of Arabidopsis plants, which metabolizes the amino acids Lys, Met, Thr, Ile and Gly, as well as a single group of "highly coordinated" genes within the branched aromatic amino acid metabolic network, which metabolizes the amino acids Trp, Phe and Tyr. These genes possess highly coordinated adjustable negative and positive expression responses to various stress cues, which apparently regulate adjustable metabolic shifts between competing branches of these networks. We also provide evidence implying that these highly coordinated genes are central to impose intra- and inter-network interactions between the Asp-family and aromatic amino acid metabolic networks as well as differential system interactions with other growth promoting and stress-associated genome-wide genes. Conclusion Our novel Gene Coordination elucidates that branched amino acid metabolic networks in plants are regulated by specific groups of highly coordinated genes that possess adjustable intra-network, inter-network and genome-wide transcriptional interactions. We also hypothesize that such transcriptional interactions enable regulatory metabolic adjustments needed for adaptation to the stresses.

Examination of Signatures of Recent Positive Selection on Genes Involved in Human Sialic Acid Biology.

Science.gov (United States)

Moon, Jiyun M; Aronoff, David M; Capra, John A; Abbot, Patrick; Rokas, Antonis

2018-03-28

Sialic acids are nine carbon sugars ubiquitously found on the surfaces of vertebrate cells and are involved in various immune response-related processes. In humans, at least 58 genes spanning diverse functions, from biosynthesis and activation to recycling and degradation, are involved in sialic acid biology. Because of their role in immunity, sialic acid biology genes have been hypothesized to exhibit elevated rates of evolutionary change. Consistent with this hypothesis, several genes involved in sialic acid biology have experienced higher rates of non-synonymous substitutions in the human lineage than their counterparts in other great apes, perhaps in response to ancient pathogens that infected hominins millions of years ago (paleopathogens). To test whether sialic acid biology genes have also experienced more recent positive selection during the evolution of the modern human lineage, reflecting adaptation to contemporary cosmopolitan or geographically-restricted pathogens, we examined whether their protein-coding regions showed evidence of recent hard and soft selective sweeps. This examination involved the calculation of four measures that quantify changes in allele frequency spectra, extent of population differentiation, and haplotype homozygosity caused by recent hard and soft selective sweeps for 55 sialic acid biology genes using publicly available whole genome sequencing data from 1,668 humans from three ethnic groups. To disentangle evidence for selection from confounding demographic effects, we compared the observed patterns in sialic acid biology genes to simulated sequences of the same length under a model of neutral evolution that takes into account human demographic history. We found that the patterns of genetic variation of most sialic acid biology genes did not significantly deviate from neutral expectations and were not significantly different among genes belonging to different functional categories. Those few sialic acid biology genes that

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

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Daniel Mihálik

2015-12-01

Full Text Available The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v and 0%–1.53% (v/v from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.

Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

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Sá-Correia Isabel

2010-10-01

Full Text Available Abstract Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5. Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to

Differences in acidity of apples are probably mainly caused by a malic acid transporter gene on LG16

NARCIS (Netherlands)

Khan, S.A.; Beekwilder, J.; Schaart, J.G.; Mumm, R.; Soriano, J.M.; Jacobsen, E.; Schouten, H.J.

2013-01-01

Acidity has profound effects on the taste of apples (Malus × domestica). Malic acid is the predominant organic acid in apples. Differences in malic acid content are caused by differences in accumulation of malic acid in the vacuole. This accumulation may be caused by a gene that is responsible for

Impact of Docosahexaenoic Acid on Gene Expression during Osteoclastogenesis in Vitro—A Comprehensive Analysis

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Ikuo Morita

2013-08-01

Full Text Available Polyunsaturated fatty acids (PUFAs, especially n-3 polyunsaturated fatty acids, docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA, are known to protect against inflammation-induced bone loss in chronic inflammatory diseases, such as rheumatoid arthritis, periodontitis and osteoporosis. We previously reported that DHA, not EPA, inhibited osteoclastogenesis induced by the receptor activator of nuclear factor-κB ligand (sRANKL in vitro. In this study, we performed gene expression analysis using microarrays to identify genes affected by the DHA treatment during osteoclastogenesis. DHA strongly inhibited osteoclastogenesis at the late stage. Among the genes upregulated by the sRANKL treatment, 4779 genes were downregulated by DHA and upregulated by the EPA treatment. Gene ontology analysis identified sets of genes related to cell motility, cell adhesion, cell-cell signaling and cell morphogenesis. Quantitative PCR analysis confirmed that DC-STAMP, an essential gene for the cell fusion process in osteoclastogenesis, and other osteoclast-related genes, such as Siglec-15, Tspan7 and Mst1r, were inhibited by DHA.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

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Meilan Xue

2012-12-01

Full Text Available Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3, which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells. Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Xue, Meilan; Ge, Yinlin; Zhang, Jinyu [Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao Shandong (China); Wang, Qing [Affiliated Hospital of Qingdao University, Qingdao Shandong (China); Hou, Lin [Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao Shandong (China)

2012-09-14

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

International Nuclear Information System (INIS)

Xue, Meilan; Ge, Yinlin; Zhang, Jinyu; Wang, Qing; Hou, Lin

2012-01-01

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings

Polymorphisms in the fatty acid desaturase genes and diet are important determinants of infant docosahexaenoic acid status

DEFF Research Database (Denmark)

Lauritzen, L.; Harsløf, L.; Larsen, L.H.

2013-01-01

Tissue docosahexaenoic acid (DHA) accretion in early infancy is supported by DHA in breast-milk and may thus decrease once complementary feeding takes over. Endogenous synthesis of DHA from alphalinolenic acid is low and polymorphisms in the genes that encodes the fatty acid desaturases (FADS) ha...

Expression and Association of SCD Gene Polymorphisms and Fatty Acid Compositions in Chicken Cross

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A. Furqon

2017-12-01

Full Text Available Stearoyl-CoA desaturase (SCD is an integral membrane protein of endoplasmic reticulum (ER that catalyzes the rate limiting step in the monounsaturated fatty acids from saturated fatty acids. Selection for fatty acids traits based on molecular marker assisted selection is needed to increase a value of chicken meat. This study was designed to analyze expression and associations of SCD gene polymorphisms with fatty acid traits in F2 kampung-broiler chicken cross. A total of 62 F2 kampung-broiler chicken cross (29 males and 33 females were used in this study. Fatty acid traits were measured at 26 weeks of age. Samples were divided into two groups based on fatty acid traits (the highest and the lowest. Primers in exon 2 region were designed from the genomic chicken sequence. The SNP g.37284A>G was detected and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method was then used to genotype. The expression of SCD gene was analyzed using quantitative real time PCR (qRT-PCR. The result showed that there were three genotypes (AA, AG, and GG found in this study. The SCD|AciI polymorphism was significantly associated with palmitoleic acid (C16:1, fatty acids total and saturated fatty acid in 26 weeks old of F2 kampung-broiler chicken cross (P<0.05. The SCD gene was expressed for polyunsaturated fatty acids in liver tissue in two groups of chickens. In conclusion, the SCD gene could be a candidate gene that affects fatty acids traits in F2 kampung-broiler chicken cross.

Role of NPR1 dependent and NPR1 independent genes in response to Salicylic acid

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Neha Agarwal

2017-10-01

Full Text Available NPR1 (Nonexpressor of pathogenesis-related gene is a transcription coactivator and central regulator of systemic acquired resistance (SAR pathway. It controls wide range of pathogenesis related genes involved in various defense responses, acts by sensing SAR signal molecule, Salicylic acid (SA. Mutation in NPR1 results in increased susceptibility to pathogen infection and less expression of pathogenesis related genes. The present study aimed to identify the role of NPR1 in gene expression after the Salicylic acid induction. For this RNA-seq was performed in Arabidopsis thaliana Col-0 and npr1-1 in response to Salicylic acid. RNA-seq analysis revealed a total of 3811 differentially expressed gene in which 2109 genes are up-regulated and 1702 genes are down-regulated. We have divided these genes in 6 categories SA induced (SI, SA repressed (SR, NPR1 dependent SI (ND-SI, NPR1 dependent SR (ND-SR, NPR1 independent SI (NI-SI, NPR1 independent SR (NI-SR. Further, Gene ontology and MapMan pathway analysis of differentially expressed genes suggested variety of biological processes and metabolic pathways that are enriched during SAR defense pathway. These results contribute to shed light on importance of both NPR1-dependent (ND and NPR1-independent (NI gene acting downstream to Salicylic acid induction in SAR pathway. The present study aimed to identify the role of NPR1 in gene expression after the Salicylic acid induction.

Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

Energy Technology Data Exchange (ETDEWEB)

Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

1991-09-11

The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

Case Report: Genetic analysis and anesthetic management of a child with Niemann-Pick disease Type A [version 1; referees: 2 approved

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Priti G. Dalal

2015-12-01

Full Text Available A 14-month-old child, recently diagnosed with Niemann-Pick disease type A, presented for a laparoscopic placement of a gastrostomy tube under general anesthesia. The disease was confirmed and further characterized by genetic testing, which revealed evidence of the presence of two known pathogenic mutations in the SMPD1 gene, and enzyme studies showed a corresponding very low level of enzymatic activity of acidic sphingomyelinase. The anesthetic management involved strategies to manage an anticipated difficult intubation and avoid post-operative ventilation.

Biocomputional construction of a gene network under acid stress in Synechocystis sp. PCC 6803.

Science.gov (United States)

Li, Yi; Rao, Nini; Yang, Feng; Zhang, Ying; Yang, Yang; Liu, Han-ming; Guo, Fengbiao; Huang, Jian

2014-01-01

Acid stress is one of the most serious threats that cyanobacteria have to face, and it has an impact at all levels from genome to phenotype. However, very little is known about the detailed response mechanism to acid stress in this species. We present here a general analysis of the gene regulatory network of Synechocystis sp. PCC 6803 in response to acid stress using comparative genome analysis and biocomputational prediction. In this study, we collected 85 genes and used them as an initial template to predict new genes through co-regulation, protein-protein interactions and the phylogenetic profile, and 179 new genes were obtained to form a complete template. In addition, we found that 11 enriched pathways such as glycolysis are closely related to the acid stress response. Finally, we constructed a regulatory network for the intricate relationship of these genes and summarize the key steps in response to acid stress. This is the first time a bioinformatic approach has been taken systematically to gene interactions in cyanobacteria and the elaboration of their cell metabolism and regulatory pathways under acid stress, which is more efficient than a traditional experimental study. The results also provide theoretical support for similar research into environmental stresses in cyanobacteria and possible industrial applications. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Polymorphisms in fatty acid metabolism-related genes are associated with colorectal cancer risk

DEFF Research Database (Denmark)

Hoeft, B.; Linseisen, J.; Beckmann, L.

2010-01-01

as contributing factor to colon carcinogenesis. We examined the association between genetic variability in 43 fatty acid metabolism-related genes and colorectal risk in 1225 CRC cases and 2032 controls participating in the European Prospective Investigation into Cancer and Nutrition study. Three hundred......Colorectal cancer (CRC) is the third most common malignant tumor and the fourth leading cause of cancer death worldwide. The crucial role of fatty acids for a number of important biological processes suggests a more in-depth analysis of inter-individual differences in fatty acid metabolizing genes...... variants with CRC risk. Our results support the key role of prostanoid signaling in colon carcinogenesis and suggest a relevance of genetic variation in fatty acid metabolism-related genes and CRC risk....

Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

DEFF Research Database (Denmark)

Nielsen, Peter E

2010-01-01

Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference...

Effect of gene transfer of Chlorella vulgaris n-3 fatty acid desaturase ...

African Journals Online (AJOL)

Chlorella vulgaris had the gene of n-3 fatty acid desaturase (CvFad3) which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or to convert n-6 to n-3 PUFAs. The objective of this study was to examine whether the CvFad3 gene from C. vulgaris can be functionally expressed in mammalian cells and ...

Effects of oils rich in linoleic and α-linolenic acids on fatty acid profile and gene expression in goat meat.

Science.gov (United States)

Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

2014-09-24

Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression.

OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH

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Alimuddin Alimuddin

2008-12-01

Full Text Available Eicosapentaenoic acid (EPA, 20:5n-3 and docosahexaenoic acid (DHA, 22:6n-3 have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3 rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD, Δ5-desaturase-like (OmΔ5FAD and elongase-like (MELO encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou were individually transferred into zebrafish (Danio rerio as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05 than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05 than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05 than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism ofSphingomyelinase-Induced Hot-Cold Hemolysis

DEFF Research Database (Denmark)

Montes, Ruth; Lopez, David; Sot, Jesus

2008-01-01

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 °C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 °C. The mechanism of this phenomenon is not understood. PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa......) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR2 activity. After......-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR2 demonstrates the presence of ceramide-rich domains that are rigid at 4 °C but fluid at 37 °C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally...

Effects of Long Chain Fatty Acid Synthesis and Associated Gene Expression in Microalga Tetraselmis sp.

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T. Catalina Adarme-Vega

2014-06-01

Full Text Available With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA to C20:4 eicosatetraenoic acid (ETA, correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding β-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4, but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt.

Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

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Baiquan Ma

2015-11-01

Full Text Available A gene encoding aluminum-activated malate transporter (ALMT was previously reported as a candidate for the locus controlling acidity in apple ( × Borkh.. In this study, we found that apple genes can be divided into three families and the gene belongs to the family. Duplication of genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the gene and its homologs in the family and only the gene is significantly associated with malic acid content. The locus consists of two alleles, and . resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the locus. This suggests that the gene is not the only genetic determinant of fruit acidity in apple.

A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans

DEFF Research Database (Denmark)

Brophy, Patrick D.; Rasmussen, Maria; Parida, Mrutyunjaya

2017-01-01

investigations have identified several gene variants that cause RA, including EYA1, LHX1, and WT1 However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we...... in humans....

Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

Science.gov (United States)

Wang, Fei; Wang, Yitong; Wang, Hui; Shao, Naimin; Chen, Yuanyuan; Cheng, Yiyun

2014-11-01

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

Science.gov (United States)

Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

2015-01-01

Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

Sphingolipid metabolism correlates with cerebrospinal fluid Beta amyloid levels in Alzheimer's disease.

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Alfred N Fonteh

Full Text Available Sphingolipids are important in many brain functions but their role in Alzheimer's disease (AD is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but

Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

Science.gov (United States)

O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

2015-08-03

The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon.

Science.gov (United States)

Kouzai, Yusuke; Kimura, Mamiko; Yamanaka, Yurie; Watanabe, Megumi; Matsui, Hidenori; Yamamoto, Mikihiro; Ichinose, Yuki; Toyoda, Kazuhiro; Onda, Yoshihiko; Mochida, Keiichi; Noutoshi, Yoshiteru

2016-03-02

Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. We propose that the Brachypodium immune

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

Science.gov (United States)

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Codon usage and amino acid usage influence genes expression level.

Science.gov (United States)

Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

2018-02-01

Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

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

African Journals Online (AJOL)

Citric acid is an important organic acid, multifunctional with a wide array of uses. The objectives of this study were the isolation and selection strains of the genus Aspergillus, investigating the solubilization of phosphate of these isolates, verifying the expression rate of genes involved in the identification of isolates, and ...

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

DEFF Research Database (Denmark)

Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

2009-01-01

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

Science.gov (United States)

Assier, E; Bouzinba-Segard, H; Stolzenberg, M C; Stephens, R; Bardos, J; Freemont, P; Charron, D; Trowsdale, J; Rich, T

1999-04-16

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae.

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Terabayashi, Yasunobu; Sano, Motoaki; Yamane, Noriko; Marui, Junichiro; Tamano, Koichi; Sagara, Junichi; Dohmoto, Mitsuko; Oda, Ken; Ohshima, Eiji; Tachibana, Kuniharu; Higa, Yoshitaka; Ohashi, Shinichi; Koike, Hideaki; Machida, Masayuki

2010-12-01

Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism. Copyright © 2010 Elsevier Inc. All rights reserved.

Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes

NARCIS (Netherlands)

Caarls, Lotte; van der Does, Adriana; Hickman, Richard; Jansen, Wouter; van Verk, Marcel; Proietti, Silvia; Lorenzo, Oscar; Solano, Roberto; Pieterse, Corné M J; Van Wees, Saskia C M

2017-01-01

Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the

Metabolism of very long-chain Fatty acids: genes and pathophysiology.

Science.gov (United States)

Sassa, Takayuki; Kihara, Akio

2014-02-01

Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology.

A co-expression gene network associated with developmental regulation of apple fruit acidity.

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Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Xu, Kenong

2015-08-01

Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'.

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

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Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

Protection of the female reproductive system from natural and artificial insults

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Tilly, Jonathan L.; Kolesnick, Richard N.

2010-12-14

Described are methods for protecting the female reproductive system against natural and artificial insults by administering to women a composition comprising an agent that antagonizes one or more acid sphingomyelinase (ASMase) gene products. Specifically, methods disclosed herein serve to protect women's germline from damage resulting from cancer therapy regimens including chemotherapy or radiotherapy. In one aspect, the method preserves, enhances, or revives ovarian function in women, by administering to women a composition containing sphingosine-1-phosphate, or an analog thereof. Also disclosed are methods to prevent or ameliorate menopausal syndromes and to improve in vitro fertilization techniques.

Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.

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Stoffel, Wilhelm; Jenke, Britta; Holz, Barbara; Binczek, Erika; Günter, Robert Heinz; Knifka, Jutta; Koebke, Jürgen; Niehoff, Anja

2007-07-01

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3(-/-) mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3(-/-) mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia.

Neutral Sphingomyelinase (SMPD3) Deficiency Causes a Novel Form of Chondrodysplasia and Dwarfism That Is Rescued by Col2A1-Driven smpd3 Transgene Expression

Science.gov (United States)

Stoffel, Wilhelm; Jenke, Britta; Holz, Barbara; Binczek, Erika; Günter, Robert Heinz; Knifka, Jutta; Koebke, Jürgen; Niehoff, Anja

2007-01-01

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3−/− mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3−/− mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia. PMID:17591962

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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Zhimin Dai

Full Text Available Biological nitrogen fixation is an essential function of acid mine drainage (AMD microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

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Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community. PMID:24498417

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

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Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

2016-01-01

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Differential Gene Expression of Longan Under Simulated Acid Rain Stress.

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Zheng, Shan; Pan, Tengfei; Ma, Cuilan; Qiu, Dongliang

2017-05-01

Differential gene expression profile was studied in Dimocarpus longan Lour. in response to treatments of simulated acid rain with pH 2.5, 3.5, and a control (pH 5.6) using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Results showed that mRNA differential display conditions were optimized to find an expressed sequence tag (EST) related with acid rain stress. The potential encoding products had 80% similarity with a transcription initiation factor IIF of Gossypium raimondii and 81% similarity with a protein product of Theobroma cacao. This fragment is the transcription factor activated by second messenger substances in longan leaves after signal perception of acid rain.

Fatty acid CoA ligase-4 gene polymorphism influences fatty acid metabolism in metabolic syndrome, but not in depression.

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Zeman, Miroslav; Vecka, Marek; Jáchymová, Marie; Jirák, Roman; Tvrzická, Eva; Stanková, Barbora; Zák, Ales

2009-04-01

The composition of polyunsaturated fatty acids (PUFAs) in cell membranes and body tissues is altered in metabolic syndrome (MetS) and depressive disorder (DD). Within the cell, fatty acid coenzyme A (CoA) ligases (FACLs) activate PUFAs by esterifying with CoA. The FACL4 isoform prefers PUFAs (arachidonic and eicosapentaenoic acid) as substrates, and the FACL4 gene is mapped to Xq23. We have analyzed the association between the common single nucleotide polymorphism (SNP) (rs1324805, C to T substitution) in the first intron of the FACL4 gene and MetS or DD. The study included 113 healthy subjects (54 Males/59 Females), 56 MetS patients (34M/22F) and 41 DD patients (7M/34F). In MetS group, T-carriers and patients with CC or C0 (CC/C0) genotype did not differ in the values of metabolic indices of MetS and M/F ratio. Nevertheless, in comparison with CC/C0, the T-allele carriers were characterized by enhanced unfavorable changes in fatty acid metabolism typical for MetS: higher content of dihomogammalinolenic acid (P phosphatidylcholine (PC) (P = 0.052), lower index of Delta5 desaturation (P insulin, conjugated dienes and index of insulin resistance, but showed no significant association with the studied SNP. The present study shows that the common SNP (C to T substitution) in the first intron of the FACL4 gene is associated with altered FA composition of plasma phosphatidylcholines in patients with MetS.

Systematic study of association of four GABAergic genes: glutamic acid decarboxylase 1 gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor 1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray.

Science.gov (United States)

Zhao, Xu; Qin, Shengying; Shi, Yongyong; Zhang, Aiping; Zhang, Jing; Bian, Li; Wan, Chunling; Feng, Guoyin; Gu, Niufan; Zhang, Guangqi; He, Guang; He, Lin

2007-07-01

Several studies have suggested the dysfunction of the GABAergic system as a risk factor in the pathogenesis of schizophrenia. In the present study, case-control association analysis was conducted in four GABAergic genes: two glutamic acid decarboxylase genes (GAD1 and GAD2), a GABA(A) receptor subunit beta2 gene (GABRB2) and a GABA(B) receptor 1 gene (GABBR1). Using a universal DNA microarray procedure we genotyped a total of 20 SNPs on the above four genes in a study involving 292 patients and 286 controls of Chinese descent. Statistically significant differences were observed in the allelic frequencies of the rs187269C/T polymorphism in the GABRB2 gene (P=0.0450, chi(2)=12.40, OR=1.65) and the -292A/C polymorphism in the GAD1 gene (P=0.0450, chi(2)=14.64 OR=1.77). In addition, using an electrophoretic mobility shift assay (EMSA), we discovered differences in the U251 nuclear protein binding to oligonucleotides representing the -292 SNP on the GAD1 gene, which suggests that the -292C allele has reduced transcription factor binding efficiency compared with the 292A allele. Using the multifactor-dimensionality reduction method (MDR), we found that the interactions among the rs187269C/T polymorphism in the GABRB2 gene, the -243A/G polymorphism in the GAD2 gene and the 27379C/T and 661C/T polymorphisms in the GAD1 gene revealed a significant association with schizophrenia (Pschizophrenia in the Chinese population.

Acid detergent lignin, lodging resistance index, and expression of the caffeic acid O-methyltransferase gene in brown midrib-12 sudangrass.

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Li, Yuan; Liu, Guibo; Li, Jun; You, Yongliang; Zhao, Haiming; Liang, Huan; Mao, Peisheng

2015-09-01

Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg(-1)) was significantly (P bmr-12 mutants (0.29) was significantly (P bmr-12 materials (r = -0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

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Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

2016-01-01

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

Memory responses of jasmonic acid-associated Arabidopsis genes to a repeated dehydration stress.

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Liu, Ning; Staswick, Paul E; Avramova, Zoya

2016-11-01

Dehydration stress activates numerous genes co-regulated by diverse signaling pathways. Upon repeated exposures, however, a subset of these genes does not respond maintaining instead transcription at their initial pre-stressed levels ('revised-response' genes). Most of these genes are involved in jasmonic acid (JA) biosynthesis, JA-signaling and JA-mediated stress responses. How these JA-associated genes are regulated to provide different responses to similar dehydration stresses is an enigma. Here, we investigate molecular mechanisms that contribute to this transcriptional behavior. The memory-mechanism is stress-specific: one exposure to dehydration stress or to abscisic acid (ABA) is required to prevent transcription in the second. Both ABA-mediated and JA-mediated pathways are critical for the activation of these genes, but the two signaling pathways interact differently during a single or multiple encounters with dehydration stress. Synthesis of JA during the first (S1) but not the second dehydration stress (S2) accounts for the altered transcriptional responses. We propose a model for these memory responses, wherein lack of MYC2 and of JA synthesis in S2 is responsible for the lack of expression of downstream genes. The similar length of the memory displayed by different memory-type genes suggests biological relevance for transcriptional memory as a gene-regulating mechanism during recurring bouts of drought. © 2016 John Wiley & Sons Ltd.

Cloning and expression of cell wall acid invertase gene fragment ...

African Journals Online (AJOL)

ONOS

2010-01-25

Jan 25, 2010 ... intron. It had a high homology to previously cloned cell wall acid invertase genes in other plants by sequence .... Japan) in a final volume of 50 µl. The programs for ... The first strand of cDNA was synthesized by using SYBR ...

Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

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Shewmaker Christine K

2010-10-01

Full Text Available Abstract Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD 2 and fatty acid elongase (FAE 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general

Serum Homocysteine, Vitamin B12, Folic Acid Levels and Methylenetetrahydrofolate Reductase (MTHFR) Gene Polymorphism in Vitiligo

Science.gov (United States)

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

2012-01-01

The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo. PMID:22846211

Serum Homocysteine, Vitamin B12, Folic Acid Levels and Methylenetetrahydrofolate Reductase (MTHFR Gene Polymorphism in Vitiligo

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Ali Yasar

2012-01-01

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

Identification of Exonic Nucleotide Variants of the Gene Associated with Carcass Traits and Fatty Acid Composition in Korean Cattle

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Dong-yep Oh

2014-10-01

Full Text Available The thyroid hormone responsive protein (THRSP gene is a functional gene that can be used to indicate the fatty acid compositions. This study investigates the relationships of exonic single nucleotide polymorphisms (SNPs in the THRSP gene and fatty acid composition of muscle fat and marbling score in the 612 Korean cattle. The relationships between fatty acid composition and eight SNPs in the THRSP gene (g.78 G>A, g.173 C>T, g.184 C>T, g.190 C>A, g.194 C>T, g.277 C>G, g.283 T>G and g.290 T>G were investigated, and according to the results, two SNPs (g.78 G>A and g.184 C>T in exon 1 were associated with fatty acid composition. The GG and CC genotypes of g.78 G>A and g.184 C>T had higher unsaturated fatty acid (UFA and monounsaturated fatty acid (MUFA content (pA and g.184 C>T had significantly relationships with UFAs and MUFAs. Two SNPs in the THRSP gene affected fatty acid composition, suggesting that GG and CC genotypes and the ht1*ht1 group (Val/Ala haplotype can be markers to genetically improve the quality and flavor of beef.

, , , , , and Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

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S. H. Choi

2015-03-01

Full Text Available We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC and intramuscular preadipocytes (IPA were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS/Dulbecco’s Modified Eagle Medium (DMEM and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC or 5% FBS/DMEM (IPA with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001 carnitine palmitoyltransferase-1 beta (CPT1β gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases. Oleic and linoleic acid decreased (p = 0.001 stearoyl-CoA desaturase (SCD gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure

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Sahbaie Peyman

2007-06-01

Full Text Available Abstract Background Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. Methods Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. Results The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. Conclusion Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.

Venom of the Brazilian spider Sicarius ornatus (Araneae, Sicariidae contains active sphingomyelinase D: potential for toxicity after envenomation.

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Priscila Hess Lopes

Full Text Available BACKGROUND: The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D. We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32-35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to

Venom of the Brazilian spider Sicarius ornatus (Araneae, Sicariidae) contains active sphingomyelinase D: potential for toxicity after envenomation.

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Lopes, Priscila Hess; Bertani, Rogério; Gonçalves-de-Andrade, Rute M; Nagahama, Roberto H; van den Berg, Carmen W; Tambourgi, Denise V

2013-01-01

The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil. SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32-35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of

Quick and sensitive determination of gene expression of fatty acid ...

African Journals Online (AJOL)

User

2011-05-16

May 16, 2011 ... from fatty acid synthase (FAS) with a different glucose level in ... By using the following formula, this study was able to quantify the mRNA expression of ... hypertension, heart disease and diabetes. ... regulation of gene expression has emerged in recent ... stages of adipocyte meta-bolism are relatively well.

Quick and sensitive determination of gene expression of fatty acid ...

African Journals Online (AJOL)

Obesity results from an imbalance between energy intake and energy expenditure, which leads to a pathological accumulation of adipose tissue, but the underlying mechanism at gene level, is far from being elucidated. The objective of this study was to investigate the correlation between mRNA express from fatty acid ...

Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

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Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

2014-09-01

Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.

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Clarke, S D

2001-04-01

This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the (n-3) family, play pivotal roles as "fuel partitioners" in that they direct fatty acids away from triglyceride storage and toward oxidation, and that they enhance glucose flux to glycogen. In doing this, PUFA may protect against the adverse symptoms of the metabolic syndrome and reduce the risk of heart disease. PUFA exert their beneficial effects by up-regulating the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously down-regulating genes encoding proteins of lipid synthesis. PUFA govern oxidative gene expression by activating the transcription factor peroxisome proliferator-activated receptor alpha. PUFA suppress lipogenic gene expression by reducing the nuclear abundance and DNA-binding affinity of transcription factors responsible for imparting insulin and carbohydrate control to lipogenic and glycolytic genes. In particular, PUFA suppress the nuclear abundance and expression of sterol regulatory element binding protein-1 and reduce the DNA-binding activities of nuclear factor Y, Sp1 and possibly hepatic nuclear factor-4. Collectively, the studies discussed suggest that the fuel "repartitioning" and gene expression actions of PUFA should be considered among criteria used in defining the dietary needs of (n-6) and (n-3) and in establishing the dietary ratio of (n-6) to (n-3) needed for optimum health benefit.

Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

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Xue, L.

2018-01-01

Full Text Available Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil. Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids solubilizing phosphate. Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can solubilize P in tricalcium phosphate (TCP medium. We aimed to obtain the gene expressed in Escherichia coli, which can enhance organic acid secretion. Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5'-end of RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid secretion ability of the transformed E. coli DH5α™. Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of 233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg·l-1 acetic acid within 48 h. The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™ clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert. Conclusions. We obtained a

Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

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Ling Zhang

2014-01-01

Full Text Available The Delta-12 oleate desaturase gene (FAD2-1, which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71% and a reduction in palmitic acid (to <3% in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

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González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2016-02-01

Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid.

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Xiao, Huogen; Siddiqua, Mahbuba; Braybrook, Siobhan; Nassuth, Annette

2006-07-01

The C-repeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/ DREB1) transcription factors control an important pathway for increased freezing and drought tolerance in plants. Three CBF/DREB1-like genes, CBF 1-3, were isolated from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced proteins in V. riparia are 63-70% identical to each other and 96-98% identical to the corresponding proteins in V. vinifera. All Vitis CBF proteins are 42-51% identical to AtCBF1 and contain CBF-specific amino acid motifs, supporting their identification as CBF proteins. Grape CBF sequences are unique in that they contain 20-29 additional amino acids and three serine stretches. Agro-infiltration experiments revealed that VrCBF1b localizes to the nucleus. VrCBF1a, VrCBF1b and VvCBF1 activated a green fluorescent protein (GFP) or glucuronidase (GUS) reporter gene behind CRT-containing promoters. Expression of the endogenous CBF genes was low at ambient temperature and enhanced upon low temperature (4 degrees C) treatment, first for CBF1, followed by CBF2, and about 2 d later by CBF3. No obvious significant difference was observed between V. riparia and V. vinifera genes. The expression levels of all three CBF genes were higher in young tissues than in older tissues. CBF1, 2 and 3 transcripts also accumulated in response to drought and exogenous abscisic acid (ABA) treatment, indicating that grape contains unique CBF genes.

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

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Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon

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Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar ‘203Z’ and its near-isogenic line (NIL) ‘SW’ (in the ‘203Z’ background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening. PMID:29324867

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon.

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Lei Gao

Full Text Available Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar '203Z' and its near-isogenic line (NIL 'SW' (in the '203Z' background were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy, sucrose-phosphate synthase (SPSs, insoluble acid invertases (IAI, NAD-dependent malate dehydrogenase (NAD-cyt MDH, aluminum-activated malate transporter (ALMT, and citrate synthase (CS. This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening.

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon.

Science.gov (United States)

Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling; Liu, Wenge

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar '203Z' and its near-isogenic line (NIL) 'SW' (in the '203Z' background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening.

Cloning and expression of cell wall acid invertase gene fragment ...

African Journals Online (AJOL)

A fragment of invertase gene containing catalytic sites of cysteine was cloned from poinsettia (Euphorbia pulcherrima wild.) by using the polymerase chain reaction (PCR) method. The length of the fragment was 521 bp, encoding 173 amino acids and containing a part of open reading frames, but no intron. It had a high ...

Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

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Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

2015-12-01

Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

Effect of cerulenin on fatty acid composition and gene expression pattern of DHA-producing strain Colwellia psychrerythraea strain 34H.

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Wan, Xia; Peng, Yun-Feng; Zhou, Xue-Rong; Gong, Yang-Min; Huang, Feng-Hong; Moncalián, Gabriel

2016-02-06

Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or β-oxidation pathway were dramatically reduced at the transcriptional level. Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation.

Profiling of hepatic gene expression in rats treated with fibric acid analogs

International Nuclear Information System (INIS)

Cornwell, Paul D.; Souza, Angus T. de; Ulrich, Roger G.

2004-01-01

Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors whose ligands include fatty acids, eicosanoids and the fibrate class of drugs. In humans, fibrates are used to treat dyslipidemias. In rodents, fibrates cause peroxisome proliferation, a change that might explain the observed hepatomegaly. In this study, rats were treated with multiple dose levels of six fibric acid analogs (including fenofibrate) for up to two weeks. Pathological analysis identified hepatocellular hypertrophy as the only sign of hepatotoxicity, and only one compound at the highest dose caused any significant increase in serum ALT or AST activity. RNA profiling revealed that the expression of 1288 genes was related to dose or length of treatment and correlated with hepatocellular hypertrophy. This gene list included expression changes that were consistent with increased mitochondrial and peroxisomal β-oxidation, increased fatty acid transport, increased hepatic uptake of LDL-cholesterol, decreased hepatic uptake of glucose, decreased gluconeogenesis and decreased glycolysis. These changes are likely linked to many of the clinical benefits of fibrate drugs, including decreased serum triglycerides, decreased serum LDL-cholesterol and increased serum HDL-cholesterol. In light of the fact that all six compounds stimulated similar or identical changes in the expression of this set of 1288 genes, these results indicate that hepatomegaly is due to PPARα activation, although signaling through other receptors (e.g. PPARγ, RXR) or through non-receptor pathways cannot be excluded

Genetics Home Reference: Niemann-Pick disease

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... are compartments within cells that break down and recycle different types of molecules. Acid sphingomyelinase is responsible ... A, Brodie SE, Desnick RJ, Wasserstein MP. Natural history of Type A Niemann-Pick disease: possible endpoints ...

Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat.

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Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

2016-01-01

Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation.

Selection of reliable reference genes for gene expression studies in Trichoderma afroharzianum LTR-2 under oxalic acid stress.

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Lyu, Yuping; Wu, Xiaoqing; Ren, He; Zhou, Fangyuan; Zhou, Hongzi; Zhang, Xinjian; Yang, Hetong

2017-10-01

An appropriate reference gene is required to get reliable results from gene expression analysis by quantitative real-time reverse transcription PCR (qRT-PCR). In order to identify stable and reliable reference genes in Trichoderma afroharzianum under oxalic acid (OA) stress, six commonly used housekeeping genes, i.e., elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, actin, from the effective biocontrol isolate T. afroharzianum strain LTR-2 were tested for their expression during growth in liquid culture amended with OA. Four in silico programs (comparative ΔCt, NormFinder, geNorm and BestKeeper) were used to evaluate the expression stabilities of six candidate reference genes. The elongation factor 1 gene EF-1 was identified as the most stably expressed reference gene, and was used as the normalizer to quantify the expression level of the oxalate decarboxylase coding gene OXDC in T. afroharzianum strain LTR-2 under OA stress. The result showed that the expression of OXDC was significantly up-regulated as expected. This study provides an effective method to quantify expression changes of target genes in T. afroharzianum under OA stress. Copyright © 2017 Elsevier B.V. All rights reserved.

Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

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González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2015-01-01

Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

Increased missense mutation burden of Fatty Acid metabolism related genes in nunavik inuit population.

Science.gov (United States)

Zhou, Sirui; Xiong, Lan; Xie, Pingxing; Ambalavanan, Amirthagowri; Bourassa, Cynthia V; Dionne-Laporte, Alexandre; Spiegelman, Dan; Turcotte Gauthier, Maude; Henrion, Edouard; Diallo, Ousmane; Dion, Patrick A; Rouleau, Guy A

2015-01-01

Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism. Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals. Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians. The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit.

Interaction of Ferulic Acid with Glutathione S-Transferase and Carboxylesterase Genes in the Brown Planthopper, Nilaparvata lugens.

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Yang, Jun; Sun, Xiao-Qin; Yan, Shu-Ying; Pan, Wen-Jun; Zhang, Mao-Xin; Cai, Qing-Nian

2017-07-01

Plant phenolics are crucial defense phytochemicals against herbivores and glutathione S-transferase (GST) and carboxylesterase (CarE) in herbivorous insects are well-known detoxification enzymes for such xenobiotics. To understand relationship between a plant phenolic and herbivore GST or CarE genes, we evaluated the relationship between a rice phenolic ferulic acid and resistance to brown planthopper (BPH, Nilaparvata lugens), and investigated the interaction of ferulic acid with GST or CarE genes in BPH. The results indicate that ferulic acid content in tested rice varieties was highly associated with resistance to BPH. Bioassays using artificial diets show that the phenolic acid toxicity to BPH was dose dependent and the LC 25 and LC 50 were 5.81 and 23.30 μg/ml at 72 hr, respectively. Activities of the enzymes BPH GST and CarE were increased at concentrations below the LC 50 of ferulic acid. Moreover, low ferulic acid concentrations (gene silencing (DIGS) of GST or CarE, it was shown that suppressed expression levels of NlGSTD1, NlGSTE1 and NlCE were 14.6%-21.2%, 27.8%-34.2%, and 10.5%-19.8%, respectively. Combination of NlGSTD1, NlGSTE1 or NlCE knockdown with ferulic acid increased nymph mortality by 92.9%, 119.9%, or 124.6%, respectively. These results suggest that depletion of detoxification genes in herbivorous insects by plant-mediated RNAi technology might be a new potential resource for improving rice resistance to BPH.

Identification of Sphingomyelinase on the Surface of Chlamydia pneumoniae: Possible Role in the Entry into Its Host Cells

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Tuula A. Peñate Medina

2014-01-01

Full Text Available We have recently suggested a novel mechanism, autoendocytosis, for the entry of certain microbes into their hosts, with a key role played by the sphingomyelinase-catalyzed topical conversion of sphingomyelin to ceramide, the differences in the biophysical properties of these two lipids providing the driving force. The only requirement for such microbes to utilize this mechanism is that they should have a catalytically active SMase on their outer surface while the target cells should expose sphingomyelin in the external leaflet of their plasma membrane. In pursuit of possible microbial candidates, which could utilize this putative mechanism, we conducted a sequence similarity search for SMase. Because of the intriguing cellular and biochemical characteristics of the poorly understood entry of Chlamydia into its host cells these microbes were of particular interest. SMase activity was measured in vitro from isolated C. pneumoniae elementary bodies (EB and in the lysate from E. coli cells transfected with a plasmid expressing CPn0300 protein having sequence similarity to SMase. Finally, pretreatment of host cells with exogenous SMase resulting in loss plasma membrane sphingomyelin attenuated attachment of EB.

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

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Ferreira, A B; Oliveira, M N V de; Freitas, F S; Paiva, A D; Alfenas-Zerbini, P; Silva, D F da; Queiroz, M V de; Borges, A C; Moraes, C A de

2015-01-01

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Amino acid diversity on the basis of cytochrome b gene in Kacang and Ettawa Grade goats

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D. A. Lestari

2017-08-01

Full Text Available The objectives of study were to identify and assess the amino acid diversity of Cytochrome b (Cyt b gene, genetic marker and characteristic of specific amino acid in Kacang and Ettawa Grade goat. Nineteen heads of Kacang goat (KG and twelve heads of Ettawa Grade goat (EG were purposively sampled. The genomic DNA was isolated by Genomic DNA Mini Kit (Geneaid and amplified Cyt b using PCR method with CytbCapF and CytbCapR primers and was sequenced. The results showed that there were two specific amino acids that distinguish KG and EG goat with C. hircus and C. aegagrus and four specific amino acids that distinguish KG and EG goat with C. falconeri, but there were no specific amino acids can be used as a genetic marker to distinguish between Kacang and EG goat. In conclusion, specific amino acids in Cyt b gene can be used as a genetic marker among KG and EG goat with 3 goat others comparator.

Characterization of the ecological role of genes mediating acid resistance in Lactobacillus reuteri during colonization of the gastrointestinal tract.

Science.gov (United States)

Krumbeck, Janina A; Marsteller, Nathan L; Frese, Steven A; Peterson, Daniel A; Ramer-Tait, Amanda E; Hutkins, Robert W; Walter, Jens

2016-07-01

Rodent-derived strains of Lactobacillus reuteri densely colonize the forestomach of mice and possess several genes whose predicted functions constitute adaptations towards an acidic environment. The objective of this study was to systematically determine which genes of L. reuteri 100-23 contribute to tolerance towards host gastric acid secretion. Genes predicted to be involved in acid resistance were inactivated, and their contribution to survival under acidic conditions was confirmed in model gastric juice. Fitness of five mutants that showed impaired in vitro acid resistance were then compared through competition experiments in ex-germ-free mice that were either treated with omeprazole, a proton-pump inhibitor that suppresses acid secretion in the stomach, or left untreated. This analysis revealed that the urease cluster was the predominant factor in mediating resistance to gastric acid production. Population levels of the mutant, which were substantially decreased in untreated mice, were almost completely restored through omeprazole, demonstrating that urease production in L. reuteri is mainly devoted to overcome gastric acid. The findings provide novel information on the mechanisms by which L. reuteri colonizes its gastric niche and demonstrate that in silico gene predictions and in vitro tests have limitations for predicting the ecological functions of colonization factors in bacterial symbionts. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

International Nuclear Information System (INIS)

Zeng, Jibin; Racicott, Jesse; Morales, Carlos R.

2009-01-01

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM 2 AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

Energy Technology Data Exchange (ETDEWEB)

Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

2009-11-01

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin

Subchronic effects of valproic acid on gene expression profiles for lipid metabolism in mouse liver

International Nuclear Information System (INIS)

Lee, Min-Ho; Kim, Mingoo; Lee, Byung-Hoon; Kim, Ju-Han; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-Il; Chung, Heekyoung; Kong, Gu; Lee, Mi-Ock

2008-01-01

Valproic acid (VPA) is used clinically to treat epilepsy, however it induces hepatotoxicity such as microvesicular steatosis. Acute hepatotoxicity of VPA has been well documented by biochemical studies and microarray analysis, but little is known about the chronic effects of VPA in the liver. In the present investigation, we profiled gene expression patterns in the mouse liver after subchronic treatment with VPA. VPA was administered orally at a dose of 100 mg/kg/day or 500 mg/kg/day to ICR mice, and the livers were obtained after 1, 2, or 4 weeks. The activities of serum liver enzymes did not change, whereas triglyceride concentration increased significantly. Microarray analysis revealed that 1325 genes of a set of 32,996 individual genes were VPA responsive when examined by two-way ANOVA (P 1.5). Consistent with our previous results obtained using an acute VPA exposure model (Lee et al., Toxicol Appl Pharmacol. 220:45-59, 2007), the most significantly over-represented biological terms for these genes included lipid, fatty acid, and steroid metabolism. Biological pathway analysis suggests that the genes responsible for increased biosynthesis of cholesterol and triglyceride, and for decreased fatty acid β-oxidation contribute to the abnormalities in lipid metabolism induced by subchronic VPA treatment. A comparison of the VPA-responsive genes in the acute and subchronic models extracted 15 commonly altered genes, such as Cyp4a14 and Adpn, which may have predictive power to distinguish the mode of action of hepatotoxicants. Our data provide a better understanding of the molecular mechanisms of VPA-induced hepatotoxicity and useful information to predict steatogenic hepatotoxicity

Profiling of hepatic gene expression in rats treated with fibric acid analogs

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Cornwell, Paul D.; Souza, Angus T. de; Ulrich, Roger G

2004-05-18

Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors whose ligands include fatty acids, eicosanoids and the fibrate class of drugs. In humans, fibrates are used to treat dyslipidemias. In rodents, fibrates cause peroxisome proliferation, a change that might explain the observed hepatomegaly. In this study, rats were treated with multiple dose levels of six fibric acid analogs (including fenofibrate) for up to two weeks. Pathological analysis identified hepatocellular hypertrophy as the only sign of hepatotoxicity, and only one compound at the highest dose caused any significant increase in serum ALT or AST activity. RNA profiling revealed that the expression of 1288 genes was related to dose or length of treatment and correlated with hepatocellular hypertrophy. This gene list included expression changes that were consistent with increased mitochondrial and peroxisomal {beta}-oxidation, increased fatty acid transport, increased hepatic uptake of LDL-cholesterol, decreased hepatic uptake of glucose, decreased gluconeogenesis and decreased glycolysis. These changes are likely linked to many of the clinical benefits of fibrate drugs, including decreased serum triglycerides, decreased serum LDL-cholesterol and increased serum HDL-cholesterol. In light of the fact that all six compounds stimulated similar or identical changes in the expression of this set of 1288 genes, these results indicate that hepatomegaly is due to PPAR{alpha} activation, although signaling through other receptors (e.g. PPAR{gamma}, RXR) or through non-receptor pathways cannot be excluded.

Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

Science.gov (United States)

Pietrowski, D; Förster, M

2000-01-01

The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

Habitual dietary intake of fatty acids are associated with leptin gene expression in subcutaneous and visceral adipose tissue of patients without diabetes.

Science.gov (United States)

Rostami, Hosein; Samadi, Mohammad; Yuzbashian, Emad; Zarkesh, Maryam; Asghari, Golaleh; Hedayati, Mehdi; Daneshafrooz, Afsoon; Mirmiran, Parvin; Khalaj, Alireza

2017-11-01

The purpose of the study was to investigate the association of leptin gene expression in visceral and subcutaneous adipose tissues with habitual fatty acid intake and its subtypes in adults. Visceral and subcutaneous adipose tissues were gathered from 97 participants aged ≥ 20, who had undergone elective abdominal surgery. Dietary fatty acid intakes including total fatty acids (TFA), saturated fatty acid (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-3, n-6, and n-9 fatty acids were collected using a valid and reliable food-frequency questionnaire (FFQ). The leptin gene expression in visceral and subcutaneous adipose tissues was measured by Real-Time PCR. After controlling for body mass index (BMI) and insulin, energy-adjusted dietary intake of SFA was positively and MUFA and n-3 fatty acids were negatively associated with subcutaneous and visceral adipose tissues leptin gene expression. Besides, a significant negative association of PUFA, n-6, and n-9 fatty acids with leptin mRNA from visceral adipose tissue were observed. In order to better interpretations of the results, the participants were allocated two groups including non-obese (BMI fatty acids had a negative association with visceral leptin gene expression. Habitual intake of SFA, MUFA, and n-3 fatty acids were associated with leptin gene expression in visceral and subcutaneous adipose tissues, suggesting an important role of quality and quantity of fatty acids intake in adipose tissue to regulate leptin expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

Science.gov (United States)

Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

2017-10-01

Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene

Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

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Riahi, Reza; Wang, Shue; Long, Min; Li, Na; Chiou, Pei-Yu; Zhang, Donna D; Wong, Pak Kin

2014-04-22

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Comparison of gene expression and fatty acid profiles in concentrate and forage finished beef.

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Buchanan, J W; Garmyn, A J; Hilton, G G; VanOverbeke, D L; Duan, Q; Beitz, D C; Mateescu, R G

2013-01-01

Fatty acid profiles and intramuscular expression of genes involved in fatty acid metabolism were characterized in concentrate- (CO) and forage- (FO) based finishing systems. Intramuscular samples from the adductor were taken at slaughter from 99 heifers finished on a CO diet and 58 heifers finished on a FO diet. Strip loins were obtained at fabrication to evaluate fatty acid profiles of LM muscle for all 157 heifers by using gas chromatography fatty acid methyl ester analysis. Composition was analyzed for differences by using the General Linear Model (GLM) procedure in SAS. Differences in fatty acid profile included a greater atherogenic index, greater percentage total MUFA, decreased omega-3 to omega-6 ratio, decreased percentage total PUFA, and decreased percentage omega-3 fatty acids in CO- compared with FO-finished heifers (P0.05). Upregulation was observed for PPARγ, fatty acid synthase (FASN), and fatty acid binding protein 4 (FABP4) in FO-finished compared with CO-finished heifers in both atherogenic index categories (P<0.05). Upregulation of diglyceride acyl transferase 2 (DGAT2) was observed in FO-finished heifers with a HAI (P<0.05). Expression of steroyl Co-A desaturase (SCD) was upregulated in CO-finished heifers with a LAI, and downregulated in FO-finished heifers with a HAI (P<0.05). Expression of adiponectin (ADIPOQ) was significantly downregulated in CO-finished heifers with a HAI compared with all other categories (P<0.05). The genes identified in this study which exhibit differential regulation in response to diet or in animals with extreme fatty acid profiles may provide genetic markers for selecting desirable fatty acid profiles in future selection programs.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria

NARCIS (Netherlands)

Bron, P.A.; Benchimol, M.G.; Lambert, J.; Palumbo, E.; Deghorain, M.; Delcour, J.; Vos, de W.M.; Kleerebezem, M.; Hols, P.

2002-01-01

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection

Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

Science.gov (United States)

Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

2017-12-01

This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l -1 , monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

Directory of Open Access Journals (Sweden)

Zhiliang Yu

2015-01-01

Full Text Available Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab, L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

Genetic alterations in fatty acid transport and metabolism genes are associated with metastatic progression and poor prognosis of human cancers.

Science.gov (United States)

Nath, Aritro; Chan, Christina

2016-01-04

Reprogramming of cellular metabolism is a hallmark feature of cancer cells. While a distinct set of processes drive metastasis when compared to tumorigenesis, it is yet unclear if genetic alterations in metabolic pathways are associated with metastatic progression of human cancers. Here, we analyzed the mutation, copy number variation and gene expression patterns of a literature-derived model of metabolic genes associated with glycolysis (Warburg effect), fatty acid metabolism (lipogenesis, oxidation, lipolysis, esterification) and fatty acid uptake in >9000 primary or metastatic tumor samples from the multi-cancer TCGA datasets. Our association analysis revealed a uniform pattern of Warburg effect mutations influencing prognosis across all tumor types, while copy number alterations in the electron transport chain gene SCO2, fatty acid uptake (CAV1, CD36) and lipogenesis (PPARA, PPARD, MLXIPL) genes were enriched in metastatic tumors. Using gene expression profiles, we established a gene-signature (CAV1, CD36, MLXIPL, CPT1C, CYP2E1) that strongly associated with epithelial-mesenchymal program across multiple cancers. Moreover, stratification of samples based on the copy number or expression profiles of the genes identified in our analysis revealed a significant effect on patient survival rates, thus confirming prominent roles of fatty acid uptake and metabolism in metastatic progression and poor prognosis of human cancers.

MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

Science.gov (United States)

Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

2009-07-01

Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

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Daniel P. Dulebohn

2017-09-01

Full Text Available The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC, BBA66, and some BosR (Borreliaoxidative stress regulator-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages.

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Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-04-25

Background: An appropriate intake of omega-3 ( n -3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n -3 FAs on gene expression levels are also dose-dependent.

IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

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Zaibi, Mohamed S; Kępczyńska, Małgorzata A; Harikumar, Parvathy; Alomar, Suliman Y; Trayhurn, Paul

2018-05-15

Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1β. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm

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Rosli, Rozana; Amiruddin, Nadzirah; Ab Halim, Mohd Amin; Chan, Pek-Lan; Chan, Kuang-Lim; Azizi, Norazah; Morris, Priscilla E.; Leslie Low, Eng-Ti; Ong-Abdullah, Meilina; Sambanthamurthi, Ravigadevi; Singh, Rajinder

2018-01-01

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops. PMID:29672525

Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm.

Science.gov (United States)

Rosli, Rozana; Amiruddin, Nadzirah; Ab Halim, Mohd Amin; Chan, Pek-Lan; Chan, Kuang-Lim; Azizi, Norazah; Morris, Priscilla E; Leslie Low, Eng-Ti; Ong-Abdullah, Meilina; Sambanthamurthi, Ravigadevi; Singh, Rajinder; Murphy, Denis J

2018-01-01

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops.

Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

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Royah Vaezi

2013-12-01

Full Text Available In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4 from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15. These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA in marine microalgae.

Toxic responses of Sox2 gene in the regeneration of the earthworm Eisenia foetida exposed to Retnoic acid.

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Tao, Jing; Rong, Wei; Diao, Xiaoping; Zhou, Hailong

2018-01-01

Exogenous retinoic acid delays and disturbs the regeneration of Eisenia foetida. The stem cell pluripotency factor, Sox2, can play a crucial role in cell reprogramming and dedifferentiation. In this study, we compared the regeneration of Eisenia foetida in different segments after amputation and the effects of retinoic acid on the regeneration of different segments. The results showed that the regeneration speed of the head and tail was slightly faster than the middle part, and retinoic acid disrupted and delayed the regeneration of the earthworm. The qRT-PCR and Western blot analysis showed that the expression of the Sox2 gene and Sox2 protein was highest on the seventh day in different segments (pregeneration of earthworms and the formation of blastema are related to the expression of the Sox2 gene and protein. Retinoic acid delays and interferes with the regeneration of the earthworm by affecting the expression levels of the Sox2 gene and protein. Copyright © 2017 Elsevier Inc. All rights reserved.

[Cloning and gene expression in lactic acid bacteria].

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Bondarenko, V M; Beliavskaia, V A

2000-01-01

The possibility of using the genera Lactobacillus and Lactococcus as vector representatives is widely discussed at present. The prospects of the construction of recombinant bacteria are closely connected with the solution of a number of problems: the level of the transcription of cloned genes, the effectiveness of the translation of heterologous mRNA, the stability of protein with respect to bacterial intracellular proteases, the method by protein molecules leave the cell (by secretion or as the result of lysis). To prevent segregation instability, the construction of vector molecules on the basis of stable cryptic plasmids found in wild strains of lactic acid bacteria was proposed. High copying plasmids with low molecular weight were detected in L. plantarum and L. pentosus strains. Several plasmids with molecular weights of 1.7, 1.8 and 2.3 kb were isolated from bacterial cells to be used as the basis for the construction of vector molecules. Genes of chloramphenicol- and erythromycin-resistance from Staphylococcus aureus plasmids were used as marker genes ensuring cell transformation. The vector plasmids thus constructed exhibited high transformation activity in the electroporation of different strains, including L. casei, L. plantarum, L. acidophilus, L. fermentum and L. brevis which could be classified with the replicons of a wide circle of hosts. But the use of these plasmids was limited due to the risk of the uncontrolled dissemination of recombinant plasmids. L. acidophilus were also found to have strictly specific plasmids with good prospects of being used as the basis for the creation of vectors, incapable of dissemination. In addition to the search of strain-specific plasmids, incapable of uncontrolled gene transmission, the use of chromosome-integrated heterologous genes is recommended in cloning to ensure the maximum safety.

Analysis of the porcine APOA2 gene expression in liver, polymorphism identification and association with fatty acid composition traits

NARCIS (Netherlands)

Ballester, M.; Revilla, M.; Puig-Oliveras, A.; Marchesi, J.A.; Castello, A.; Corominas, J.; Fernandez, A.I.; Folch, J.M.

2016-01-01

APOA2 is a protein implicated in triglyceride, fatty acid and glucose metabolism. In pigs, the APOA2 gene is located on pig chromosome 4 (SSC4) in a QTL region affecting fatty acid composition, fatness and growth traits. In this study, we evaluated APOA2 as a candidate gene for meat quality traits

Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

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Igor R. Costa

2014-12-01

Full Text Available Essential amino acids (EAA consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS and betaine-homocysteine S-methyltransferase (BHMT diverged from the expected Tree of Life (ToL relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

Impact of methoxyacetic acid on mouse Leydig cell gene expression

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Waxman David J

2010-06-01

Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

Polymorphisms in Fatty Acid Desaturase (FADS) Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA) Supplementation

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Cormier, Hubert; Rudkowska, Iwona; Thifault, Elisabeth; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2013-01-01

Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM). Polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS) and serum fatty acid composition. Objective: To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG) and fasting insulin (FI) responses following a 6-week n-3 polyunsaturated fatty acids (PUFA) supplementation. Methods: 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g–2.2 g of EPA + 1.1 g of DHA). Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03) was performed. Results: Carriers of the minor allele for rs482548 (FADS2) had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008). For FI levels, a genotype effect was observed with one SNP (rs174456). For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively). Conclusion: Results suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation. PMID:24705214

Polymorphisms in Fatty Acid Desaturase (FADS Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA Supplementation

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Patrick Couture

2013-09-01

Full Text Available Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM. Polymorphisms (SNPs in the fatty acid desaturase (FADS gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS and serum fatty acid composition. Objective: To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG and fasting insulin (FI responses following a 6-week n-3 polyunsaturated fatty acids (PUFA supplementation. Methods: 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g–2.2 g of EPA + 1.1 g of DHA. Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03 was performed. Results: Carriers of the minor allele for rs482548 (FADS2 had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008. For FI levels, a genotype effect was observed with one SNP (rs174456. For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively. Conclusion: Results suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation.

Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

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Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

2014-01-01

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

Amino acid-substituted gemini surfactant-based nanoparticles as safe and versatile gene delivery agents.

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Singh, Jagbir; Yang, Peng; Michel, Deborah; Verrall, Ronald E; Foldvari, Marianna; Badea, Ildiko

2011-05-01

Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

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Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

2014-01-01

In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

Effect of α-linolenic acid and DHA intake on lipogenesis and gene expression involved in fatty acid metabolism in growing-finishing pigs.

Science.gov (United States)

De Tonnac, A; Labussière, E; Vincent, A; Mourot, J

2016-07-01

The regulation of lipogenesis mechanisms related to consumption of n-3 PUFA is poorly understood. The aim of the present study was to find out whether α-linolenic acid (ALA) or DHA uptake can have an effect on activities and gene expressions of enzymes involved in lipid metabolism in the liver, subcutaneous adipose tissue and longissimus dorsi (LD) muscle of growing-finishing pigs. Six groups of ten pigs received one of six experimental diets supplemented with rapeseed oil in the control diet, extruded linseed, microalgae or a mixture of both to implement different levels of ALA and DHA with the same content in total n-3. Results were analysed for linear and quadratic effects of DHA intake. The results showed that activities of malic enzyme (ME) and fatty acid synthase (FAS) decreased linearly in the liver with dietary DHA. Although the expression of the genes of these enzymes and their activities were poorly correlated, ME and FAS expressions also decreased linearly with DHA intake. The intake of DHA down-regulates the expressions of other genes involved in fatty acid (FA) metabolism in some tissues of pigs, such as fatty acid desaturase 2 and sterol-regulatory element binding transcription factor 1 in the liver and 2,4-dienoyl CoA reductase 2 in the LD muscle. FA oxidation in the LD muscle and FA synthesis decreased in the liver with increasing amount of dietary DHA, whereas a retroconversion of DHA into EPA seems to be set up in this last tissue.

Horizontal gene transfer in an acid mine drainage microbial community.

Science.gov (United States)

Guo, Jiangtao; Wang, Qi; Wang, Xiaoqi; Wang, Fumeng; Yao, Jinxian; Zhu, Huaiqiu

2015-07-04

Horizontal gene transfer (HGT) has been widely identified in complete prokaryotic genomes. However, the roles of HGT among members of a microbial community and in evolution remain largely unknown. With the emergence of metagenomics, it is nontrivial to investigate such horizontal flow of genetic materials among members in a microbial community from the natural environment. Because of the lack of suitable methods for metagenomics gene transfer detection, microorganisms from a low-complexity community acid mine drainage (AMD) with near-complete genomes were used to detect possible gene transfer events and suggest the biological significance. Using the annotation of coding regions by the current tools, a phylogenetic approach, and an approximately unbiased test, we found that HGTs in AMD organisms are not rare, and we predicted 119 putative transferred genes. Among them, 14 HGT events were determined to be transfer events among the AMD members. Further analysis of the 14 transferred genes revealed that the HGT events affected the functional evolution of archaea or bacteria in AMD, and it probably shaped the community structure, such as the dominance of G-plasma in archaea in AMD through HGT. Our study provides a novel insight into HGT events among microorganisms in natural communities. The interconnectedness between HGT and community evolution is essential to understand microbial community formation and development.

Gene Transcription and Virulence Potential of Listeria monocytogenes Strains After Exposure to Acidic and NaCl Stress

DEFF Research Database (Denmark)

Olesen, Inger; Vogensen, Finn Kvist; Jespersen, Lene

2009-01-01

transcription were observed both after exposure to shock (six genes) and after long-term adaptation to stress (18 genes). In the shock experiments, a transient induction of clpC and clpE was seen for both strains, while transient induction of sigB, inlA, and inlB was observed for strain 4140 only; actA was only...... induced in EGD-e after NaCl shock. The longterm stress experiments were included to imitate the stress conditions encountered by L. monocytogenes when present in food products. Long-term adaptation of EGD-e to acidic stress induced transcription of iap and repressed flaA, while genes related to stress......Gene transcription and virulence potential of two strains of Listeria monocytogenes, EGD-e and 4140, were compared by quantitative real-time polymerase chain reaction and in a Caco-2 in vitro model after exposure to acidic (pH 5.5) and NaCl (4.5% w=v) stress. Strain-dependent differences in gene...

Transcriptomic profiling of linolenic acid-responsive genes in ROS signalling from RNA-seq data in Arabidopsis

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Capilla eMata-Pérez

2015-03-01

Full Text Available Linolenic acid (Ln released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA. The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signalling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2 fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs and Allene oxide cyclases (AOCs. In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding, such as WRKY, JAZ, MYC and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signalling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1, methionine sulfoxide reductase (MSR and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signalling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity and especially in stresses mediated by ROS.

The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils.

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Ryan, P R; Tyerman, S D; Sasaki, T; Furuichi, T; Yamamoto, Y; Zhang, W H; Delhaize, E

2011-01-01

Acid soils restrict plant production around the world. One of the major limitations to plant growth on acid soils is the prevalence of soluble aluminium (Al(3+)) ions which can inhibit root growth at micromolar concentrations. Species that show a natural resistance to Al(3+) toxicity perform better on acid soils. Our understanding of the physiology of Al(3+) resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. Fourteen genes from seven different species are known to contribute to Al(3+) tolerance and resistance and several additional candidates have been identified. Some of these genes account for genotypic variation within species and others do not. One mechanism of resistance which has now been identified in a range of species relies on the efflux of organic anions such as malate and citrate from roots. The genes controlling this trait are members of the ALMT and MATE families which encode membrane proteins that facilitate organic anion efflux across the plasma membrane. Identification of these and other resistance genes provides opportunities for enhancing the Al(3+) resistance of plants by marker-assisted breeding and through biotechnology. Most attempts to enhance Al(3+) resistance in plants with genetic engineering have targeted genes that are induced by Al(3+) stress or that are likely to increase organic anion efflux. In the latter case, studies have either enhanced organic anion synthesis or increased organic anion transport across the plasma membrane. Recent developments in this area are summarized and the structure-function of the TaALMT1 protein from wheat is discussed.

Short- and long-term changes in sugarbeet (Beta vulgaris L. gene expression due to postharvest jasmonic acid treatment - Data

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Lucilene Silva de Oliveira

2017-04-01

Full Text Available Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L. root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr, Swiss-Prot, Clusters of Orthologous Groups (COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG protein databases, and unigene annotations with Gene Ontology (GO terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017 [1]. Public dissemination of this dataset will allow further analyses of the data.

Amino acid transporter genes are essential for FLO11-dependent and FLO11-independent biofilm formation and invasive growth in Saccharomyces cerevisiae.

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Rasmus Torbensen

Full Text Available Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype. Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3(647::CWNKNPLSSIN allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3(647::CWNKNPLSSIN-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.

Liver Gene Expression Profiles of Rats Treated with Clofibric Acid

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Michel, Cécile; Desdouets, Chantal; Sacre-Salem, Béatrice; Gautier, Jean-Charles; Roberts, Ruth; Boitier, Eric

2003-01-01

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor α, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci. PMID:14633594

Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes.

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Caarls, Lotte; Van der Does, Dieuwertje; Hickman, Richard; Jansen, Wouter; Verk, Marcel C Van; Proietti, Silvia; Lorenzo, Oscar; Solano, Roberto; Pieterse, Corné M J; Van Wees, Saskia C M

2017-02-01

Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the ERF transcription factor family typically bind to GCC-box motifs in the promoters of JA- and ethylene-responsive genes, thereby positively or negatively regulating their expression. The GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Here, we investigated whether SA-induced ERF-type transcriptional repressors, which may compete with JA-induced ERF-type activators for binding at the GCC-box, play a role in SA/JA antagonism. We selected ERFs that are transcriptionally induced by SA and/or possess an EAR transcriptional repressor motif. Several of the 16 ERFs tested suppressed JA-dependent gene expression, as revealed by enhanced JA-induced PDF1.2 or VSP2 expression levels in the corresponding erf mutants, while others were involved in activation of these genes. However, SA could antagonize JA-induced PDF1.2 or VSP2 in all erf mutants, suggesting that the tested ERF transcriptional repressors are not required for SA/JA cross-talk. Moreover, a mutant in the co-repressor TOPLESS, that showed reduction in repression of JA signaling, still displayed SA-mediated antagonism of PDF1.2 and VSP2. Collectively, these results suggest that SA-regulated ERF transcriptional repressors are not essential for antagonism of JA-responsive gene expression by SA. We further show that de novo SA-induced protein synthesis is required for suppression of JA-induced PDF1.2, pointing to SA-stimulated production of an as yet unknown protein that suppresses JA-induced transcription. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Role of arachidonic acid metabolism in transcriptional induction of tumor necrosis factor gene expression by phorbol ester

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Horiguchi, J.; Spriggs, D.; Imamura, K.; Stone, R.; Luebbers, R.; Kufe, D.

1989-01-01

The treatment of human HL-60 promyelocytic leukemia cells with 12-0 tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcripts. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNS mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase, A/sub 2/ blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B/sub 4/. In contrast, the cyclooxygenase metabolite prostaglandin E/sub 2/ inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B/sub 4/ and prostaglandin E/sub 2/.

An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

International Nuclear Information System (INIS)

Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi; Horiuchi, Hiroyuki; Ohta, Akinori

2010-01-01

Research highlights: → POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. → Deletion of POR1 caused growth defects on fatty acids. → Δpor1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in β-oxidation and peroxisome proliferation by oleate was distinctly diminished in the Δpor1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

DEFF Research Database (Denmark)

Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

2017-01-01

The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide–oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described...

Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza

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Zhichao eXu

2016-02-01

Full Text Available Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and 4 alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that 6 candidate cytochrome P450s and 5 candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation.

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Hsueh, Yi-Huang; Huang, Kai-Yao; Kunene, Sikhumbuzo Charles; Lee, Tzong-Yi

2017-12-07

Poly-γ-glutamic acid (γ-PGA) is a biodegradable biopolymer produced by several bacteria, including Bacillus subtilis and other Bacillus species; it has good biocompatibility, is non-toxic, and has various potential biological applications in the food, pharmaceutical, cosmetic, and other industries. In this review, we have described the mechanisms of γ-PGA synthesis and gene regulation, its role in fermentation, and the phylogenetic relationships among various pgsBCAE , a biosynthesis gene cluster of γ-PGA, and pgdS , a degradation gene of γ-PGA. We also discuss potential applications of γ-PGA and highlight the established genetic recombinant bacterial strains that produce high levels of γ-PGA, which can be useful for large-scale γ-PGA production.

Synthesis and evaluation of a glutamic acid-modified hPAMAM complex as a promising versatile gene carrier.

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Hemmati, Mohammad; Kazemi, Bahram; Najafi, Farhood; Zarebkohan, Amir; Shirkoohi, Reza

2016-01-01

Hyperbranched poly(amidoamine) (HPAMAM), structurally analogous to polyamidoamine dendrimer (PAMAM) dendrimers, has been suggested to be an effective carrier for gene delivery. In the present study, glutamic acid-modified hPAMAM was developed as a novel non-viral gene carrier for the first time. The hPAMAM was synthesized by using a modified one-pot method. DNA was found to be bound to hPAMAM at different weight ratios (WhPAMAM/WDNA). The resulting HPAMAM-Glu20 was able to efficiently protect the encapsulated-DNA against degradation for over 2 h. In addition to low cytotoxicity, the transfection efficiency of hPAMAM-Glu20 represented much higher (p glutamic amino acid (Glu)-based gene delivery is an economical, effective and biocompatible method.

Dynamic changes in prefrontal cortex gene expression following lysergic acid diethylamide administration.

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Nichols, Charles D; Garcia, Efrain E; Sanders-Bush, Elaine

2003-03-17

Lysergic acid diethylamide (LSD) is a psychoactive drug that transiently alters human perception, behavior, and mood at extremely low doses. Certain aspects of the behavior elicited by acute doses of LSD closely resemble symptoms of mental disorders such as schizophrenia. Characterizing gene expression profiles after LSD will be important for understanding how it alters behavior, and will lead to novel insights into disorders, such as schizophrenia, whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs. We previously identified a small collection of genes within the rat prefrontal cortex that respond to LSD. Many of the products of these genes are involved in the process of synaptic plasticity. In the current report, we present a detailed analysis of the expression of these genes within the brain using RNase protection analysis. We find that the gene response to LSD is quite dynamic. The expression of some genes increases rapidly and decreases rapidly, while other genes change more gradually. Dose-response studies show two classes of expression; gene expression maximally stimulated at lower doses, versus gene expression that continues to rise at the higher doses. The role of the 5-HT(1A) and 5-HT(2A) receptor in mediating the increases in gene expression was examined in a series of experiments using receptor specific antagonists. Most expression increases were due to activation of the 5-HT(2A) receptor, however expression of two genes had neither a 5-HT(1A) nor a 5-HT(2A) receptor component.

Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

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Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

2002-10-15

The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

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Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

2003-03-04

The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

Polymorphism in the fatty acid desaturase genes and diet are important determinants of infant n-3 fatty acid status

DEFF Research Database (Denmark)

Harsløf, L.B.S.; Larsen, L.H.; Ritz, C.

and polymorphism in the genes that encodes the fatty acid desaturases (FADS) has little effect on DHA-status in adults. It is however unclear to what extent endogenous DHA-synthesis contributes to infant DHA-status. Aim: To investigate the role of diet and FADS polymorphism on DHA-status at 9 months and 3 years...... breastfeeding was obtained by questionnaires and fish intake was assessed by 7-day pre-coded food diaries. Results: FADS-genotype, breastfeeding, and fish intake were found to explain 25% of the variation in infant RBC DHA-status (mean±SD: 6.6±1.9% of the fatty acids (FA%)). Breastfeeding was the most important......Background and objectives: Tissue docosahexaenoic acid (DHA) accretion in early infancy has been shown to be supported by the DHA-content of breast-milk and thus may decrease once complementary feeding takes over. Endogenous synthesis of DHA from alpha-linolenic acid has been shown to be very low...

Differential expression of fatty acid transporters and fatty acid synthesis-related genes in crop tissues of male and female pigeons (Columba livia domestica) during incubation and chick rearing.

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Xie, Peng; Wang, Xue-Ping; Bu, Zhu; Zou, Xiao-Ting

2017-10-01

1. The growth performance of squabs reared solely by male or female parent pigeons was measured, and the changes of lipid content of crop milk and the expression profiles of genes potentially involved in lipid accumulation by crop tissues of parent pigeons were evaluated during incubation and chick rearing. 2. Squabs increased in body weight during 25 d of rearing, whereas both male and female pigeons lost weight after finishing rearing chicks, and the weight loss of male pigeons was significantly greater than that of female parent pigeons. Lipid content of crop milk from both parent pigeons gradually decreased to the crude fat level in the formulated diet after 10 d (R10) of chick rearing. 3. The gene expression of fatty acid translocase (FAT/CD36), fatty acid-binding protein 5 (EFABP) and acyl-CoA-binding protein (ACBP) in male pigeon crop tissue were the greatest at 17 d (I17) of incubation. In female pigeons, FAT/CD36 expression was the highest at I14, and both EFABP and ACBP expression peaked at I14 and R7. The expression of acetyl-CoA carboxylase and fatty acid synthase in male pigeons reached the maximum level at R1, while they peaked at I14 and I17, respectively in female pigeons. The gene expression of peroxisome proliferators-activated receptor-gamma (PPARγ) was the greatest at I17 in the male, while it was at I14 in the female. However, no regular changing pattern was found in PPARα gene expression in male pigeons. 4. These results indicated that male and female pigeons may make different contributions in rearing squabs. The gene expression study suggested that fatty acids used in lipid biosynthesis of crop milk probably originated from both exogenous supply and de novo synthesis. The sex of the parent pigeon affected the lipid content of crop milk and the expression profiles of genes involved in fatty acid transportation and lipogenesis.

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis.

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Nair, Ramesh B; Bastress, Kristen L; Ruegger, Max O; Denault, Jeff W; Chapple, Clint

2004-02-01

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.

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Jensen, Kristian K; Previs, Stephen F; Zhu, Lei; Herath, Kithsiri; Wang, Sheng-Ping; Bhat, Gowri; Hu, Guanghui; Miller, Paul L; McLaren, David G; Shin, Myung K; Vogt, Thomas F; Wang, Liangsu; Wong, Kenny K; Roddy, Thomas P; Johns, Douglas G; Hubbard, Brian K

2012-01-15

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.

Dietary supplementation with arginine and glutamic acid enhances key lipogenic gene expression in growing pigs.

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Hu, C J; Jiang, Q Y; Zhang, T; Yin, Y L; Li, F N; Su, J Y; Wu, G Y; Kong, X F

2017-12-01

Our previous study showed dietary supplementation with Arg and Glu increased intramuscular fat deposition and decreased back fat thickness in pigs, suggesting that the genes involved in lipid metabolism might be regulated differently in muscle and s.c. adipose (SA) tissues. Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were randomly assigned to 1 of 5 treatment groups (castrated male to female ratio = 1:1). Pigs in the control group were fed a basic diet, and those in experimental groups were fed the basic diet supplemented with 2.05% alanine (isonitrogenous group), 1.00% arginine (Arg group), 1.00% glutamic acid + 1.44% alanine (Glu group), or 1.00% arginine + 1.00% glutamic acid (Arg+Glu group). Fatty acid percentages and mRNA expression levels of the genes involved in lipid metabolism in muscle and SA tissues were examined. The percentages of C14:0 and C16:0 in the SA tissue of Glu group pigs and C14:0 in the longissimus dorsi (LD) muscle of Glu and Arg+Glu groups decreased ( acid synthase in the Arg+Glu group was more upregulated ( < 0.05) than that of the Arg group. An increase in the mRNA level of in the biceps femoris muscle was also observed in the Arg+Glu group ( < 0.05) compared with the basic diet and isonitrogenous groups. Collectively, these findings suggest that dietary supplementation with Arg and Glu upregulates the expression of genes involved in adipogenesis in muscle tissues and lipolysis in SA tissues.

A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

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Wen-Bin Xue

2016-07-01

Full Text Available The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA. To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively.

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

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Gonzalez, Lauren E; Keller, Kristen; Chan, Karen X; Gessel, Megan M; Thines, Bryan C

2017-07-17

The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes

DEFF Research Database (Denmark)

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke

2017-01-01

of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression...... antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act...... as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract....

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

Science.gov (United States)

Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

2015-09-30

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.

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Ijssennagger, Noortje; Janssen, Aafke W F; Milona, Alexandra; Ramos Pittol, José M; Hollman, Danielle A A; Mokry, Michal; Betzel, Bark; Berends, Frits J; Janssen, Ignace M; van Mil, Saskia W C; Kersten, Sander

2016-05-01

The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

NARCIS (Netherlands)

Martens, Thomas F.; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E.; Van Steenbergen, Mies J.; Demeester, Jo; De Smedt, Stefaan C.; Braeckmans, Kevin

2015-01-01

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or

Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

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Schlotz, Nina; Roulin, Anne; Ebert, Dieter; Martin-Creuzburg, Dominik

2016-11-01

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

International Nuclear Information System (INIS)

Bacha, N.; Dao, H.P.; Mathieu, F.; Liboz, T.; Lebrihi, A.; Atoui, A.; O'Callaghan, J.; Dobson, A.D.W.; Puel, O.

2008-01-01

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298 bp polyketide synthase gene ''aomsas'' has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40-56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant ''aomsas'' of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant aomsas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aomsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone. (author)

Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation

Science.gov (United States)

Hsueh, Yi-Huang; Huang, Kai-Yao; Kunene, Sikhumbuzo Charles; Lee, Tzong-Yi

2017-01-01

Poly-γ-glutamic acid (γ-PGA) is a biodegradable biopolymer produced by several bacteria, including Bacillus subtilis and other Bacillus species; it has good biocompatibility, is non-toxic, and has various potential biological applications in the food, pharmaceutical, cosmetic, and other industries. In this review, we have described the mechanisms of γ-PGA synthesis and gene regulation, its role in fermentation, and the phylogenetic relationships among various pgsBCAE, a biosynthesis gene cluster of γ-PGA, and pgdS, a degradation gene of γ-PGA. We also discuss potential applications of γ-PGA and highlight the established genetic recombinant bacterial strains that produce high levels of γ-PGA, which can be useful for large-scale γ-PGA production. PMID:29215550

Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation

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Yi-Huang Hsueh

2017-12-01

Full Text Available Poly-γ-glutamic acid (γ-PGA is a biodegradable biopolymer produced by several bacteria, including Bacillus subtilis and other Bacillus species; it has good biocompatibility, is non-toxic, and has various potential biological applications in the food, pharmaceutical, cosmetic, and other industries. In this review, we have described the mechanisms of γ-PGA synthesis and gene regulation, its role in fermentation, and the phylogenetic relationships among various pgsBCAE, a biosynthesis gene cluster of γ-PGA, and pgdS, a degradation gene of γ-PGA. We also discuss potential applications of γ-PGA and highlight the established genetic recombinant bacterial strains that produce high levels of γ-PGA, which can be useful for large-scale γ-PGA production.

QTL for phytosterol and sinapate ester content in Brassica napus L. collocate with the two erucic acid genes

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Amar, Samija; Ecke, Wolfgang; Becker, Heiko C.

2008-01-01

Improving oil and protein quality for food and feed purposes is an important goal in rapeseed (Brassica napus L.) breeding programs. Rapeseed contains phytosterols, used to enrich food products, and sinapate esters, which are limiting the utilization of rapeseed proteins in the feed industry. Increasing the phytosterol content of oil and lowering sinapate ester content of meal could increase the value of the oilseed rape crop. The objective of the present study was to identify quantitative trait loci (QTL) for phytosterol and sinapate ester content in a winter rapeseed population of 148 doubled haploid lines, previously found to have a large variation for these two traits. This population also segregated for the two erucic acid genes. A close negative correlation was found between erucic acid and phytosterol content (Spearman’s rank correlation, rs = −0.80**). For total phytosterol content, three QTL were detected, explaining 60% of the genetic variance. The two QTL with the strongest additive effects were mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. For sinapate ester content four QTL were detected, explaining 53% of the genetic variance. Again, a close negative correlation was found between erucic acid and sinapate ester content (rs = −0.66**) and the QTL with the strongest additive effects mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. The results suggests, that there is a pleiotropic effect of the two erucic acid genes on phytosterol and sinapate ester content; the effect of the alleles for low erucic acid content is to increase phytosterol and sinapate ester content. Possible reasons for this are discussed based on known biosynthetic pathways. Electronic supplementary material The online version of this article (doi:10.1007/s00122-008-0734-2) contains supplementary material, which is available to authorized users. PMID:18335203

Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis

International Nuclear Information System (INIS)

Das, Kaberi P.; Wood, Carmen R.; Lin, Mimi T.; Starkov, Anatoly A.; Lau, Christopher; Wallace, Kendall B.; Corton, J. Christopher; Abbott, Barbara D.

2017-01-01

Highlights: • Structurally diverse PFAAs induced fatty liver and increased TG accumulation in mouse. • Genes of lipid synthesis and degradation were increased after exposure to PFAAs. • PFAAs did not inhibit either mitochondrial fatty acid transport or β-oxidation directly. - Abstract: Persistent presence of perfluoroalkyl acids (PFAAs) in the environment is due to their extensive use in industrial and consumer products, and their slow decay. Biochemical tests in rodent demonstrated that these chemicals are potent modifiers of lipid metabolism and cause hepatocellular steatosis. However, the molecular mechanism of PFAAs interference with lipid metabolism remains to be elucidated. Currently, two major hypotheses are that PFAAs interfere with mitochondrial beta-oxidation of fatty acids and/or they affect the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in liver. To determine the ability of structurally-diverse PFAAs to cause steatosis, as well as to understand the underlying molecular mechanisms, wild-type (WT) and PPARα-null mice were treated with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), or perfluorohexane sulfonate (PFHxS), by oral gavage for 7 days, and their effects were compared to that of PPARα agonist WY-14643 (WY), which does not cause steatosis. Increases in liver weight and cell size, and decreases in DNA content per mg of liver, were observed for all compounds in WT mice, and were also seen in PPARα-null mice for PFOA, PFNA, and PFHxS, but not for WY. In Oil Red O stained sections, WT liver showed increased lipid accumulation in all treatment groups, whereas in PPARα-null livers, accumulation was observed after PFNA and PFHxS treatment, adding to the burden of steatosis observed in control (untreated) PPARα-null mice. Liver triglyceride (TG) levels were elevated in WT mice by all PFAAs and in PPARα-null mice only by PFNA. In vitro β-oxidation of palmitoyl carnitine by isolated rat

DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

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Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

2015-01-01

Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

DNA methylation perturbations in genes involved in polyunsaturated fatty acid biosynthesis associated with depression and suicide risk

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Fatemeh eHaghighi

2015-04-01

Full Text Available Polyunsaturated fatty acid (PUFA status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6 and α-linolenic acid (ALA, 18:3n-3. We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD with (n=22 and without (n=39 history of suicide attempt, and age- and sex-matched healthy volunteers (n=59. Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1 and 2 (Fads2, and elongation of very long chain fatty acids protein 5 (Elovl5, were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator (LASSO logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

Insights into the Indian peanut genotypes for ahFAD2 gene polymorphism regulating its oleic and linoleic acid fluxes

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Bhagwat Nawade

2016-08-01

Full Text Available In peanut (Arachis hypogaea L., the customization of fatty acid profile is an evolving area to fulfil the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and ‘SunOleic’‒ a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89% than in ssp. fastigiata (17%. This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39 respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

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Rivero Montserrat

2011-04-01

Full Text Available Abstract Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA, a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A or by oral gavage (Group B, supplemented just during suckling (Group C and control animals (Group D was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix. Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf, tissue inhibitor of metalloproteinase 1 (Timp1, galanin (Gal, synaptotagmin 1 (Syt1, growth factor receptor bound protein 2 (Grb2, actin gamma 2 (Actg2 and smooth muscle alpha actin (Acta2, as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis.

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Elashry, Abdelnaser; Okumoto, Sakiko; Siddique, Shahid; Koch, Wolfgang; Kreil, David P; Bohlmann, Holger

2013-09-01

The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

L-lactic acid production by Aspergillus brasiliensis overexpressing the heterologous ldha gene from Rhizopus oryzae.

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Liaud, Nadège; Rosso, Marie-Noëlle; Fabre, Nicolas; Crapart, Sylvaine; Herpoël-Gimbert, Isabelle; Sigoillot, Jean-Claude; Raouche, Sana; Levasseur, Anthony

2015-05-03

Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production. To investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 × 10(-2) U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan. We obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed

Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment

International Nuclear Information System (INIS)

Pezzoli, Daniele; Candiani, Gabriele

2013-01-01

Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

The histone-like protein HU has a role in gene expression during the acid adaptation response in Helicobacter pylori.

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Álvarez, Alhejandra; Toledo, Héctor

2017-08-01

Gastritis, ulcers, and gastric malignancy have been linked to human gastric epithelial colonization by Helicobacter pylori. Characterization of the mechanisms by which H. pylori adapts to the human stomach environment is of crucial importance to understand H. pylori pathogenesis. In an effort to extend our knowledge of these mechanisms, we used proteomic analysis and qRT-PCR to characterize the role of the histone-like protein HU in the response of H. pylori to low pH. Proteomic analysis revealed that genes involved in chemotaxis, oxidative stress, or metabolism are under control of the HU protein. Also, expression of the virulence factors Ggt and NapA is affected by the null mutation of hup gene both at neutral and acid pH, as evidenced by qRT-PCR analysis. Those results showed that H. pylori gene expression is altered by shift to low pH, thus confirming that acid exposure leads to profound changes in genomic expression, and suggest that the HU protein is a regulator that may help the bacterium adapt to the acid stress. In accordance with previous reports, we found that the HU protein participates in gene expression regulation when the microorganism is exposed to acid stress. Such transcriptional regulation underlies protein accumulation in the H. pylori cell. © 2017 John Wiley & Sons Ltd.

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.

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Grandvalet, Cosette; Assad-García, Juan Simón; Chu-Ky, Son; Tollot, Marie; Guzzo, Jean; Gresti, Joseph; Tourdot-Maréchal, Raphaëlle

2008-09-01

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

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Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

2005-07-15

A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

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

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Emilia Wilmowicz

2016-03-01

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

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

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Maliza, Rita; Fujiwara, Ken; Azuma, Morio; Kikuchi, Motoshi; Yashiro, Takashi

2017-06-29

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10 -6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

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Wang, Renxue; Liu, Lin; Sheps, Jonathan A; Forrest, Dana; Hofmann, Alan F; Hagey, Lee R; Ling, Victor

2013-08-15

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

Directory of Open Access Journals (Sweden)

Lei Anping

2012-03-01

Full Text Available Abstract Background Biofuel has been the focus of intensive global research over the past few years. The development of 4th generation biofuel production (algae-to-biofuels based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation. Results We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP, 3-ketoacyl-ACP-synthase (KAS, and acyl-ACP thioesterase (FATA gene expression had significant correlations with monounsaturated FA (MUFA synthesis and polyunsaturated FA (PUFA synthesis. Conclusions We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.

UV-C-Induced alleviation of transcriptional gene silencing through plant–plant communication: Key roles of jasmonic acid and salicylic acid pathways

Energy Technology Data Exchange (ETDEWEB)

Xu, Wei; Wang, Ting [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Xu, Shaoxin [School of physics and materials science, Anhui University, Hefei, Anhui, 230601 (China); Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Bian, Po, E-mail: bianpo@ipp.ac.cn [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China)

2016-08-15

Highlights: • Transcriptional gene silencing (TGS) in plants can be epigenetically alleviated by volatile signals from UV-C- irradiated neighboring plants. • Alleviation of TGS can be induced by UV-C irradiation through plant–plant–plant communication. • JA and SA signals take part in interplant communication for alleviation of TGS. - Abstract: Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant–plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant–plant and plant–plant–plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant–plant and plant–plant–plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant–plant–plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA.

UV-C-Induced alleviation of transcriptional gene silencing through plant–plant communication: Key roles of jasmonic acid and salicylic acid pathways

International Nuclear Information System (INIS)

Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

2016-01-01

Highlights: • Transcriptional gene silencing (TGS) in plants can be epigenetically alleviated by volatile signals from UV-C- irradiated neighboring plants. • Alleviation of TGS can be induced by UV-C irradiation through plant–plant–plant communication. • JA and SA signals take part in interplant communication for alleviation of TGS. - Abstract: Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant–plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant–plant and plant–plant–plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant–plant and plant–plant–plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant–plant–plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA.

Clomipramine counteracts lipid raft disturbance due to short-term muscle disuse.

Science.gov (United States)

Bryndina, Irina G; Shalagina, Maria N; Sekunov, Alexey V; Zefirov, Andrei L; Petrov, Alexey M

2018-01-18

Disuse-induced skeletal muscle dysfunction is a serious consequence of long-term spaceflight, numerous diseases and conditions for which treatment possibilities are still strictly limited. We have previously shown that acute hindlimb suspension (HS)-mediated disuse disrupts membrane lipid rafts in the unloaded muscle. Here, we investigated whether pretreatment of rats with the inhibitor of acid sphingomyelinase, clomipramine (1.25mg/g/day, intramuscularly, for 5days before HS), is able to hinder the loss in lipid raft integrity in response to 12h of HS. Clomipramine pretreatment significantly counteracted the decrease in labeling of the plasma membranes with lipid raft markers (fluorescent cholera toxin B subunit and bodipy-GM1-ganglioside) specifically in the junctional regions of the suspended soleus muscle. This was associated with: a) enhancing raft disrupting potential of exogenous sphingomyelinase in the junctional membranes; b) prevention of both ceramide accumulation and cholesterol loss; c) prevention of decline in nicotinic acetylcholine receptor labeling in the unloaded muscle. Our data suggest that sphingomyelinase-mediated raft disturbance serves as one of the earlier events in HS effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Fatty acid cosubstrates provide β-oxidation precursors for rhamnolipid biosynthesis in Pseudomonas aeruginosa, as evidenced by isotope tracing and gene expression assays.

Science.gov (United States)

Zhang, Lin; Veres-Schalnat, Tracey A; Somogyi, Arpad; Pemberton, Jeanne E; Maier, Raina M

2012-12-01

Rhamnolipids have multiple potential applications as "green" surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d(35) as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C(10) lipid chain were observed for octadecanoic acid-d(35) treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and β-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200- to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3- to 4-fold of the fadA β-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and β-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.

Global gene expression changes in human urothelial cells exposed to low-level monomethylarsonous acid

International Nuclear Information System (INIS)

Medeiros, Matthew; Zheng, Xinghui; Novak, Petr; Wnek, Shawn M.; Chyan, Vivian; Escudero-Lourdes, Claudia; Gandolfi, A. Jay

2012-01-01

Highlights: ► Chronic exposure to 50 nM monomethylarsonous acid in UROtsa was investigated. ► At 3 months of exposure substantial changes were observed in gene expression. ► Notable changes occurred in mitogenic signaling, stress, immune and inflammatory responses. ► Gene expression changes correlate with phenotypic changes from previous studies. -- Abstract: Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa) at concentrations 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A microarray analysis was performed to assess the transcriptional changes in UROtsa during the critical window of chronic 50 nM MMA(III) exposure that leads to transformation at 3 months of exposure. The analysis revealed only minor changes in gene expression at 1 and 2 months of exposure, contrasting with substantial changes observed at 3 months of exposure. The gene expression changes at 3 months were analyzed showing distinct alterations in biological processes and pathways such as a response to oxidative stress, enhanced cell proliferation, anti-apoptosis, MAPK signaling, as well as inflammation. Twelve genes selected as markers of these particular biological processes were used to validate the microarray and these genes showed a time-dependent changes at 1 and 2 months of exposure, with the most substantial changes occurring at 3 months of exposure. These results indicate that there is a strong association between the acquired phenotypic changes that occur with chronic MMA(III) exposure and the observed gene expression patterns that are indicative of a malignant transformation. Although the substantial changes that occur at 3 months of exposure may be a consequence of transformation, there are common occurrences of altered

Ceramide formation is involved in Lactobacillus acidophilus-induced IFN-beta response in dendritic cells

DEFF Research Database (Denmark)

Fuglsang, Eva; Henningsen, Louise; Frøkiær, Hanne

of sphingomyelin to ceramide by acid sphingomyelinase (ASMase) at the outer leaflet of the PM is a key event in endocytosis of gram-positive Lactobacillus acidophilus (L. acidophilus) and the subsequent induction of IFN-beta in DCs and, as the gram-negative Escherichia coli (E. coli) does not induce appreciable...

Detection of Promyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARα Fusion Gene with Functionalized Graphene Oxide

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Hongwei Wang

2013-06-01

Full Text Available An attempt was made to use functionalized graphene oxide (GO to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene, a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable.

Deregulation of PPARβ/δ target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment

Science.gov (United States)

Finkernagel, Florian; Lieber, Sonja; Schnitzer, Evelyn; Legrand, Nathalie; Schober, Yvonne; Nockher, W. Andreas; Toth, Philipp M.; Diederich, Wibke E.; Nist, Andrea; Stiewe, Thorsten; Wagner, Uwe; Reinartz, Silke; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma. PMID:25968567

Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

NARCIS (Netherlands)

Mach Casellas, N.; Jacobs, A.A.A.; Kruijt, L.; Baal, van J.; Smits, M.C.J.

2014-01-01

The aim of this study was to determine the effects of unprotected dietary unsaturated fatty acids (UFA) from different plant oils on gene expression in the mammary gland of grazing dairy cows. Milk composition and gene expression in the mammary gland tissue were evaluated in grazing dairy cows

First study of correlation between oleic acid content and SAD gene polymorphism in olive oil samples through statistical and bayesian modeling analyses.

Science.gov (United States)

Ben Ayed, Rayda; Ennouri, Karim; Ercişli, Sezai; Ben Hlima, Hajer; Hanana, Mohsen; Smaoui, Slim; Rebai, Ahmed; Moreau, Fabienne

2018-04-10

Virgin olive oil is appreciated for its particular aroma and taste and is recognized worldwide for its nutritional value and health benefits. The olive oil contains a vast range of healthy compounds such as monounsaturated free fatty acids, especially, oleic acid. The SAD.1 polymorphism localized in the Stearoyl-acyl carrier protein desaturase gene (SAD) was genotyped and showed that it is associated with the oleic acid composition of olive oil samples. However, the effect of polymorphisms in fatty acid-related genes on olive oil monounsaturated and saturated fatty acids distribution in the Tunisian olive oil varieties is not understood. Seventeen Tunisian olive-tree varieties were selected for fatty acid content analysis by gas chromatography. The association of SAD.1 genotypes with the fatty acids composition was studied by statistical and Bayesian modeling analyses. Fatty acid content analysis showed interestingly that some Tunisian virgin olive oil varieties could be classified as a functional food and nutraceuticals due to their particular richness in oleic acid. In fact, the TT-SAD.1 genotype was found to be associated with a higher proportion of mono-unsaturated fatty acids (MUFA), mainly oleic acid (C18:1) (r = - 0.79, p SAD.1 association with the oleic acid composition of olive oil was identified among the studied varieties. This correlation fluctuated between studied varieties, which might elucidate variability in lipidic composition among them and therefore reflecting genetic diversity through differences in gene expression and biochemical pathways. SAD locus would represent an excellent marker for identifying interesting amongst virgin olive oil lipidic composition.

UV-C-Induced alleviation of transcriptional gene silencing through plant-plant communication: Key roles of jasmonic acid and salicylic acid pathways.

Science.gov (United States)

Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

2016-08-01

Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant-plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant-plant and plant-plant-plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant-plant and plant-plant-plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant-plant-plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA. Copyright © 2016 Elsevier B.V. All rights reserved.

Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew.

Directory of Open Access Journals (Sweden)

Chloé Marchive

Full Text Available Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

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Wei Hu

Full Text Available The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

Science.gov (United States)

Hu, Wei; Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger

Science.gov (United States)

Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research. PMID:28650980

[Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].

Science.gov (United States)

Chakravorty, S; Sarkar, S; Gachhui, R

2015-01-01

The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.

Differences in growth, fillet quality, and fatty acid metabolism-related gene expression between juvenile male and female rainbow trout.

Science.gov (United States)

Manor, Meghan L; Cleveland, Beth M; Kenney, P Brett; Yao, Jianbo; Leeds, Tim

2015-04-01

Sexual maturation occurs at the expense of stored energy and nutrients, including lipids; however, little is known regarding sex effects on nutrient regulatory mechanisms in rainbow trout prior to maturity. Thirty-two, 14-month-old, male and female rainbow trout were sampled for growth, carcass yield, fillet composition, and gene expression of liver, white muscle, and visceral adipose tissue. Growth parameters, including gonadosomatic index, were not affected by sex. Females had higher percent separable muscle yield, but there were no sex effects on fillet proximate composition. Fillet shear force indicated females produce firmer fillets than males. Male livers had greater expression of three cofactors within the mTOR signaling pathway that act to inhibit TORC1 assembly; mo25, rictor, and pras40. Male liver also exhibited increased expression of β-oxidation genes cpt1b and ehhadh. These findings are indicative of increased mitochondrial β-oxidation in male liver. Females exhibited increased expression of the mTOR cofactor raptor in white muscle and had higher expression levels of several genes within the fatty acid synthesis pathway, including gpat, srebp1, scd1, and cd36. Female muscle also had increased expression of β-oxidation genes cpt1d and cpt2. Increased expression of both fatty acid synthesis and β-oxidation genes suggests female muscle may have greater fatty acid turnover. Differences between sexes were primarily associated with variation of gene expression within the mTOR signaling pathway. Overall, data suggest there is differential regulation of gene expression in male and female rainbow trout tissues prior to the onset of sexual maturity that may lead to nutrient repartitioning during maturation.

Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress.

Science.gov (United States)

Zhang, Luan; Xiong, Zhi-ting; Xu, Zhong-rui; Liu, Chen; Cai, Shen-wen

2014-06-01

The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3µM Cu(2+) (control) and 10µM Cu(2+) for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation. Copyright © 2014 Elsevier Inc. All rights reserved.

Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701.

Science.gov (United States)

Yuhara, Kahori; Yonehara, Hiromi; Hattori, Takasumi; Kobayashi, Keiichi; Kirimura, Kohtaro

2015-11-01

trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980. © 2015 FEBS.

Omega-3 Fatty Acid Enriched Chevon (Goat Meat Lowers Plasma Cholesterol Levels and Alters Gene Expressions in Rats

Directory of Open Access Journals (Sweden)

Mahdi Ebrahimi

2014-01-01

Full Text Available In this study, control chevon (goat meat and omega-3 fatty acid enriched chevon were obtained from goats fed a 50% oil palm frond diet and commercial goat concentrate for 100 days, respectively. Goats fed the 50% oil palm frond diet contained high amounts of α-linolenic acid (ALA in their meat compared to goats fed the control diet. The chevon was then used to prepare two types of pellets (control or enriched chevon that were then fed to twenty-male-four-month-old Sprague-Dawley rats (n=10 in each group for 12 weeks to evaluate their effects on plasma cholesterol levels, tissue fatty acids, and gene expression. There was a significant increase in ALA and docosahexaenoic acid (DHA in the muscle tissues and liver of the rats fed the enriched chevon compared with the control group. Plasma cholesterol also decreased (P<0.05 in rats fed the enriched chevon compared to the control group. The rat pellets containing enriched chevon significantly upregulated the key transcription factor PPAR-γ and downregulated SREBP-1c expression relative to the control group. The results showed that the omega-3 fatty acid enriched chevon increased the omega-3 fatty acids in the rat tissues and altered PPAR-γ and SREBP-1c genes expression.

Omega-3 fatty acid enriched chevon (goat meat) lowers plasma cholesterol levels and alters gene expressions in rats.

Science.gov (United States)

Ebrahimi, Mahdi; Rajion, Mohamed Ali; Meng, Goh Yong; Soleimani Farjam, Abdoreza

2014-01-01

In this study, control chevon (goat meat) and omega-3 fatty acid enriched chevon were obtained from goats fed a 50% oil palm frond diet and commercial goat concentrate for 100 days, respectively. Goats fed the 50% oil palm frond diet contained high amounts of α-linolenic acid (ALA) in their meat compared to goats fed the control diet. The chevon was then used to prepare two types of pellets (control or enriched chevon) that were then fed to twenty-male-four-month-old Sprague-Dawley rats (n = 10 in each group) for 12 weeks to evaluate their effects on plasma cholesterol levels, tissue fatty acids, and gene expression. There was a significant increase in ALA and docosahexaenoic acid (DHA) in the muscle tissues and liver of the rats fed the enriched chevon compared with the control group. Plasma cholesterol also decreased (P < 0.05) in rats fed the enriched chevon compared to the control group. The rat pellets containing enriched chevon significantly upregulated the key transcription factor PPAR-γ and downregulated SREBP-1c expression relative to the control group. The results showed that the omega-3 fatty acid enriched chevon increased the omega-3 fatty acids in the rat tissues and altered PPAR-γ and SREBP-1c genes expression.

Screening for Glucosyltransferase gene (gtf from exopolysaccahride producing lactic acid bacteria

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Donna M. Ariestanti

2008-04-01

Full Text Available Glucosyltransferase (GTF is an enzyme involved in exopolysaccharide (EPS polymer synthesis in microbes. One example of EPS that has been used in pharmaceutical and medical application is dextran. Dextran has been used in conjugated-drug delivery system as matrix. As a group of microbes producing EPS, lactic acid bacteria (LAB have been well reported carrying sucrase genes glucosyltransferase (gtf, as well as fructosyltransferases (ftf. In an attempt to search for novel gtf genes as the aim of this study, LAB collection isolated from local sources yielded from previous study were screened performing PCR using degenerate primers DegFor and DegRev. An approximately 660 base pairs (bp amplicons were obtained by using genomic DNAs of those LAB isolates as templates with conserved region of gtf genes catalytic domain as target. Two out of 20 LAB strains were yielded no amplicon as observed on agarose gel, while one strain exhibited non-specific amplicon DNA bands with sizes other than 660 bp. The two negative ones were isolated from soil obtained from dairy product waste field and from waste of soy sauce from previous study, while the latter was isolated from waste of soy sauce.

Cloning of D-lactate dehydrogenase genes of Lactobacillus delbrueckii subsp. bulgaricus and their roles in D-lactic acid production.

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Huang, Yanna; You, Chunping; Liu, Zhenmin

2017-07-01

Lactobacillus delbrueckii subsp. bulgaricus is a heterogenous lactic acid bacterium that converts pyruvate mainly to D-lactic acid using D-lactate dehydrogenases (D-LDHs), whose functional properties remain poorly characterized. Here, the D-LDHs genes (ldb0101, ldb0813, ldb1010, ldb1147 and ldb2021) were cloned and overexpressed in Escherichia coli JM109 from an inducible pUC18 vector, respectively, and the resulting strains were compared in terms of D-lactic acid production. The strain expressing ldb0101 and ldb1010 gene individually produced more D-lactate than other three strains. Further study revealed that Ldb0101 activity was down-regulated by the oxygen and, therefore, achieved a highest titer of D-lactate (1.94 g/L) under anaerobic condition, and introduction of ldb1010 gene enhanced D-lactate formation (0.94 and 0.85 g/L, respectively) both in aerobic and anaerobic conditions due to a relatively stable q d-lactate . Our results suggested that the enzyme Ldb0101 and Ldb1010 played a role of more importance in D-lactate formation. To the best of our knowledge, we demonstrate for the first time the roles of different D-LDH homologs from L. bulgaricus in D-lactic acid production.

Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance.

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Qin, Xiaoqiong; Zeevaart, Jan A D

2002-02-01

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.

Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Rinker, Torri E.; Baker, Scott E.

2007-01-29

Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In this pelleted form, A. niger has increased citric acid production as compared to growth in filamentous form. Chitin is a crucial component of the cell wall of filamentous fungi. Alterations in the deposition or production of chitin may have profound effects on the morphology of the organism. In order to study the role of chitin synthesis in pellet formation we have deleted a chitin synthase gene (csmA) in Aspergillus niger strain ATCC 11414 using a PCR based deletion construct. This class of chitin synthases is only found in filamentous fungi and is not present in yeasts. The csmA genes contain a myosin motor domain at the N-terminus and a chitin synthesis domain at the C-terminus. They are believed to contribute to the specialized polar growth observed in filamentous fungi that is lacking in yeasts. The csmA deletion strain (csmAΔ) was subjected to minimal media with and without osmotic stabilizers as well as tested in citric acid production media. Without osmotic stabilizers, the mutant germlings were abnormally swollen, primarily in the subapical regions, and contained large vacuoles. However, this swelling is ultimately not inhibitory to growth as the germlings are able to recover and undergo polar growth. Colony formation was largely unaffected in the absence of osmotic stabilizers. In citric acid production media csmAΔ was observed to have a 2.5 fold increase in citric acid production. The controlled expression of this class of chitin synthases may be useful for improving production of organic acids in filamentous fungi.

Mechanism analysis of acid tolerance response of bifidobacterium longum subsp. longum BBMN 68 by gene expression profile using RNA-sequencing.

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Junhua Jin

Full Text Available To analyze the mechanism of the acid tolerance response (ATR in Bifidobacterium longum subsp. longum BBMN68, we optimized the acid-adaptation condition to stimulate ATR effectively and analyzed the change of gene expression profile after acid-adaptation using high-throughput RNA-Seq. After acid-adaptation at pH 4.5 for 2 hours, the survival rate of BBMN68 at lethal pH 3.5 for 120 min was increased by 70 fold and the expression of 293 genes were upregulated by more than 2 fold, and 245 genes were downregulated by more than 2 fold. Gene expression profiling of ATR in BBMN68 suggested that, when the bacteria faced acid stress, the cells strengthened the integrity of cell wall and changed the permeability of membrane to keep the H(+ from entering. Once the H(+ entered the cytoplasm, the cells showed four main responses: First, the F(0F(1-ATPase system was initiated to discharge H(+. Second, the ability to produce NH(3 by cysteine-cystathionine-cycle was strengthened to neutralize excess H(+. Third, the cells started NER-UVR and NER-VSR systems to minimize the damage to DNA and upregulated HtpX, IbpA, and γ-glutamylcysteine production to protect proteins against damage. Fourth, the cells initiated global response signals ((pppGpp, polyP, and Sec-SRP to bring the whole cell into a state of response to the stress. The cells also secreted the quorum sensing signal (AI-2 to communicate between intraspecies cells by the cellular signal system, such as two-component systems, to improve the overall survival rate. Besides, the cells varied the pathways of producing energy by shifting to BCAA metabolism and enhanced the ability to utilize sugar to supply sufficient energy for the operation of the mechanism mentioned above. Based on these reults, it was inferred that, during industrial applications, the acid resistance of bifidobacteria could be improved by adding BCAA, γ-glutamylcysteine, cysteine, and cystathionine into the acid-stress environment.

Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

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Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

2013-11-01

Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

In Vivo Imaging of Retinoic Acid Receptor Activity using a Sodium/Iodide Symporter and Luciferase Dual Imaging Reporter Gene

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Min Kyung So

2004-07-01

Full Text Available Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL. After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.

Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

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Morral Núria

2011-01-01

Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

Construction and application of a Korean reference panel for imputing classical alleles and amino acids of human leukocyte antigen genes.

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Kim, Kwangwoo; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol

2014-01-01

Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.

Construction and application of a Korean reference panel for imputing classical alleles and amino acids of human leukocyte antigen genes.

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Kwangwoo Kim

Full Text Available Genetic variations of human leukocyte antigen (HLA genes within the major histocompatibility complex (MHC locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

DEFF Research Database (Denmark)

Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico

2013-01-01

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

Tubular urate transporter gene polymorphisms differentiate patients with gout who have normal and decreased urinary uric acid excretion.

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Torres, Rosa J; de Miguel, Eugenio; Bailén, Rebeca; Banegas, José R; Puig, Juan G

2014-09-01

Primary gout has been associated with single-nucleotide polymorphisms (SNP) in several tubular urate transporter genes. No study has assessed the association of reabsorption and secretion urate transporter gene SNP with gout in a single cohort of documented primary patients with gout carefully subclassified as normoexcretors or underexcretors. Three reabsorption SNP (SLC22A12/URAT1, SLC2A9/GLUT9, and SLC22A11/OAT4) and 2 secretion transporter SNP (SLC17A1/NPT1 and ABCG2/BRCP) were studied in 104 patients with primary gout and in 300 control subjects. The patients were subclassified into normoexcretors and underexcretors according to their serum and 24-h urinary uric acid levels under strict conditions of dietary control. Compared with control subjects, patients with gout showed different allele distributions of the 5 SNP analyzed. However, the diagnosis of underexcretor was only positively associated with the presence of the T allele of URAT1 rs11231825, the G allele of GLUT9 rs16890979, and the A allele of ABCG2 rs2231142. The association of the A allele of ABCG2 rs2231142 in normoexcretors was 10 times higher than in underexcretors. The C allele of NPT1 rs1165196 was only significantly associated with gout in patients with normal uric acid excretion. Gout with uric acid underexcretion is associated with transporter gene SNP related mainly to tubular reabsorption, whereas uric acid normoexcretion is associated only with tubular secretion SNP. This finding supports the concept of distinctive mechanisms to account for hyperuricemia in patients with gout with reduced or normal uric acid excretion.

Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15

NARCIS (Netherlands)

A.H.M. Geurts van Kessel (Ad); H. de Leeuw (H.); E.J. Dekker (Erik Jan); J.M. Rijks (Jolianne); N. Spurr (N.); A.M. Ledbetter (Andrew M.); E. Kootwijk (E.); M.J. Vaessen (Marie-Josée)

1991-01-01

textabstractA human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17)

Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

Science.gov (United States)

Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

2012-05-01

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

Arabidopsis GLUTATHIONE REDUCTASE1 plays a crucial role in leaf responses to intracellular hydrogen peroxide and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways.

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Mhamdi, Amna; Hager, Jutta; Chaouch, Sejir; Queval, Guillaume; Han, Yi; Taconnat, Ludivine; Saindrenan, Patrick; Gouia, Houda; Issakidis-Bourguet, Emmanuelle; Renou, Jean-Pierre; Noctor, Graham

2010-07-01

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H(2)O(2)) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H(2)O(2) signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H(2)O(2). Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H(2)O(2)-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H(2)O(2) availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

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Tamano, Koichi; Miura, Ai

2016-09-01

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

Science.gov (United States)

Sun, Xiaowen; Wu, Hefang; Zhao, Genhai; Li, Zhemin; Wu, Xihua; Liu, Hui; Zheng, Zhiming

2018-04-02

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

Isolation and Characterization of a Gene Specific to Lager Brewing Yeast That Encodes a Branched-Chain Amino Acid Permease

Science.gov (United States)

Kodama, Yukiko; Omura, Fumihiko; Ashikari, Toshihiko

2001-01-01

We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts. PMID:11472919

Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes.

Science.gov (United States)

Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke; Færgeman, Nils J; Kallipolitis, Birgitte H

The foodborne pathogen Listeria monocytogenes is the causative agent of the invasive disease listeriosis. Infection by L. monocytogenes involves bacterial crossing of the intestinal barrier and intracellular replication in a variety of host cells. The PrfA protein is the master regulator of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression in the intestinal environment. In the gastrointestinal phase of infection, the bacterium encounters a variety of antimicrobial agents, including medium- and long-chain free fatty acids that are commonly found in our diet and as active components of bile. Here we show that subinhibitory concentrations of specific antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A retinoic acid response element that overlaps an estrogen response element mediates multihormonal sensitivity in transcriptional activation of the lactoferrin gene.

OpenAIRE

Lee, M O; Liu, Y; Zhang, X K

1995-01-01

The lactoferrin gene is highly expressed in many different tissues, and its expression is controlled by different regulators. In this report, we have defined a retinoic acid response element (RARE) in the 5'-flanking region of the lactoferrin gene promoter. The lactoferrin-RARE is composed of two AGGTCA-like motifs arranged as a direct repeat with 1-bp spacing (DR-1). A gel retardation assay demonstrated that it bound strongly with retinoid X receptor (RXR) homodimers and RXR-retinoic acid re...

RNA Sequencing and Coexpression Analysis Reveal Key Genes Involved in α-Linolenic Acid Biosynthesis in Perilla frutescens Seed

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Tianyuan Zhang

2017-11-01

Full Text Available Perilla frutescen is used as traditional food and medicine in East Asia. Its seeds contain high levels of α-linolenic acid (ALA, which is important for health, but is scarce in our daily meals. Previous reports on RNA-seq of perilla seed had identified fatty acid (FA and triacylglycerol (TAG synthesis genes, but the underlying mechanism of ALA biosynthesis and its regulation still need to be further explored. So we conducted Illumina RNA-sequencing in seven temporal developmental stages of perilla seeds. Sequencing generated a total of 127 million clean reads, containing 15.88 Gb of valid data. The de novo assembly of sequence reads yielded 64,156 unigenes with an average length of 777 bp. A total of 39,760 unigenes were annotated and 11,693 unigenes were found to be differentially expressed in all samples. According to Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis, 486 unigenes were annotated in the “lipid metabolism” pathway. Of these, 150 unigenes were found to be involved in fatty acid (FA biosynthesis and triacylglycerol (TAG assembly in perilla seeds. A coexpression analysis showed that a total of 104 genes were highly coexpressed (r > 0.95. The coexpression network could be divided into two main subnetworks showing over expression in the medium or earlier and late phases, respectively. In order to identify the putative regulatory genes, a transcription factor (TF analysis was performed. This led to the identification of 45 gene families, mainly including the AP2-EREBP, bHLH, MYB, and NAC families, etc. After coexpression analysis of TFs with highly expression of FAD2 and FAD3 genes, 162 TFs were found to be significantly associated with two FAD genes (r > 0.95. Those TFs were predicted to be the key regulatory factors in ALA biosynthesis in perilla seed. The qRT-PCR analysis also verified the relevance of expression pattern between two FAD genes and partial candidate TFs. Although it has been reported that some TFs

Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities

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Alfa Herrera

2017-03-01

Full Text Available Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE. IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like “vegetations” composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an S. aureus virulence factor that contributes to the microorganism’s ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N, and biofilm ligase-deficient β-toxin (H162A and/or D163A on human aortic endothelial cells (HAECs and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8 from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1. HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity.

The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

Science.gov (United States)

Wathugala, Deepthi L; Hemsley, Piers A; Moffat, Caroline S; Cremelie, Pieter; Knight, Marc R; Knight, Heather

2012-07-01

• Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Overexpression of a 9-cis-Epoxycarotenoid Dioxygenase Gene in Nicotiana plumbaginifolia Increases Abscisic Acid and Phaseic Acid Levels and Enhances Drought Tolerance1

Science.gov (United States)

Qin, Xiaoqiong; Zeevaart, Jan A.D.

2002-01-01

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. PMID:11842158

Production of natural fragrance aromatic acids by coexpression of trans-anethole oxygenase and p-anisaldehyde dehydrogenase genes of Pseudomonas putida JYR-1 in Escherichia coli.

Science.gov (United States)

Han, Dongfei; Kurusarttra, Somwang; Ryu, Ji-Young; Kanaly, Robert A; Hur, Hor-Gil

2012-12-05

A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli , the engineered strain has potential to be applied in the fragrance industry.

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4α

International Nuclear Information System (INIS)

Klapper, Maja; Boehme, Mike; Nitz, Inke; Doering, Frank

2007-01-01

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4α (HNF-4α), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4α binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4α by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4α, that are both candidate genes for diabetes type 2, may be a powerful approach

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

Energy Technology Data Exchange (ETDEWEB)

Klapper, Maja [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Boehme, Mike [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Nitz, Inke [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Doering, Frank [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany)

2007-04-27

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

Science.gov (United States)

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

2013-01-01

While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

MMS sensitivity of all amino acid-requiring mutants in aspergillus and its suppression by mutations in a single gene.

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Käfer, E

1987-04-01

All available amino acid-requiring mutants of Aspergillus nidulans were found to be hypersensitive to MMS (methyl methanesulfonate) to various degrees. On MMS media, secondary mutations could be selected which suppress this MMS sensitivity but do not affect the requirement. Many such mutations were analyzed and found to be alleles of one gene, smsA (= suppressor of MMS sensitivity), which mapped distal on the right arm of chromosome V. This gene is more likely to be involved in general regulation of amino acid biosynthesis than MMS uptake, since a variety of pathway interactions were clearly modified by smsA suppressors in the absence of MMS.

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

Science.gov (United States)

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of

Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

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Abderrazak Kitsy

Full Text Available The essential branched-chain amino acids (BCAA, leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2 and branched-chain alpha keto acid dehydrogenase (Bckdha was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4 compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

International Nuclear Information System (INIS)

Wallden, Brett; Emond, Mary; Swift, Mari E; Disis, Mary L; Swisshelm, Karen

2005-01-01

The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10 -8 ), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

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Marina Kemmerer

Full Text Available AMP-activated protein kinase (AMPK maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO. The transcription factor peroxisome proliferator-activated receptor δ (PPARδ also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

Epigenetic regulation of the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs

NARCIS (Netherlands)

Corominas, J.; Marchesi, J.A.; Puig-Oliveras, A.; Revilla, M.; Estelle, J.; Alves, E.; Folch, J.M.; Ballester, M.

2015-01-01

BACKGROUND: In previous studies on an Iberian x Landrace cross, we have provided evidence that supported the porcine ELOVL6 gene as the major causative gene of the QTL on pig chromosome 8 for palmitic and palmitoleic acid contents in muscle and backfat. The single nucleotide polymorphism (SNP)

Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

Science.gov (United States)

Basyuni, M.; Wati, R.

2017-01-01

This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

DEFF Research Database (Denmark)

Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

2008-01-01

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

Transcriptomic Analysis Identifies Candidate Genes Related to Intramuscular Fat Deposition and Fatty Acid Composition in the Breast Muscle of Squabs (Columba

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Manhong Ye

2016-07-01

Full Text Available Despite the fact that squab is consumed throughout the world because of its high nutritional value and appreciated sensory attributes, aspects related to its characterization, and in particular genetic issues, have rarely been studied. In this study, meat traits in terms of pH, water-holding capacity, intramuscular fat content, and fatty acid profile of the breast muscle of squabs from two meat pigeon breeds were determined. Breed-specific differences were detected in fat-related traits of intramuscular fat content and fatty acid composition. RNA-Sequencing was applied to compare the transcriptomes of muscle and liver tissues between squabs of two breeds to identify candidate genes associated with the differences in the capacity of fat deposition. A total of 27 differentially expressed genes assigned to pathways of lipid metabolism were identified, of which, six genes belonged to the peroxisome proliferator-activated receptor signaling pathway along with four other genes. Our results confirmed in part previous reports in livestock and provided also a number of genes which had not been related to fat deposition so far. These genes can serve as a basis for further investigations to screen markers closely associated with intramuscular fat content and fatty acid composition in squabs. The data from this study were deposited in the National Center for Biotechnology Information (NCBI’s Sequence Read Archive under the accession numbers SRX1680021 and SRX1680022. This is the first transcriptome analysis of the muscle and liver tissue in Columba using next generation sequencing technology. Data provided here are of potential value to dissect functional genes influencing fat deposition in squabs.

Whey protein hydrolysate and branched-chain amino acids downregulate inflammation-related genes in vascular endothelial cells.

Science.gov (United States)

Da Silva, Marine S; Bigo, Cyril; Barbier, Olivier; Rudkowska, Iwona

2017-02-01

A recent review of clinical studies reports that dairy products may improve inflammation, a key etiologic cardiovascular disease risk factor. Yet the impact of dairy proteins on inflammatory markers is controversial and could be mediated by a differential impact of whey proteins and caseins. In this study, we hypothesized that whey proteins may have a greater anti-inflammatory effect than caseins. A model of human umbilical vein endothelial cells, with or without TNF-α stimulation, was used to investigate the effect of several dairy protein compounds on inflammation. Specifically, the impact of whey proteins either isolate or hydrolysate, caseins, and their amino acids on expression of TNF, VCAM-1, SOD2, and eNOS was examined. After a 24-hour incubation period, whey protein hydrolysate, leucine, isoleucine, and valine attenuated the TNF-α-induced endothelial inflammation by normalizing TNF and eNOS gene expression. This effect was not observed in unstimulated cells. Oppositely, caseins, a whey protein/casein mixture (1:4 w/w), and glutamine aggravated the TNF-α-induced TNF and SOD2 gene expression. Yet caseins and whey protein/casein mixture decreased VCAM-1 expression in both unstimulated and stimulated human umbilical vein endothelial cells. Measurement of TNF-α in cell supernatants by immunoassay substantiates gene expression data without reaching statistical significance. Taken together, this study showed that whey proteins and their major amino acids normalize TNF-α-induced proinflammatory gene expression in endothelial cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Centimetre-scale vertical variability of phenoxy acid herbicide mineralization potential in aquifer sediment relates to the abundance of tfdA genes

DEFF Research Database (Denmark)

Pazarbasi, Meric Batioglu; Bælum, Jacob; Johnsen, Anders R.

2012-01-01

sampled just below the groundwater table. Mineralization of 2,4-D and MCPA was fastest in sediment samples taken close to the groundwater table, whereas only minor mineralization of MCPP was seen. Considerable variability was exhibited at increasing aquifer depth, more so with 2,4-D than with MCPA...... are known to be involved in the metabolism of phenoxy acid herbicides. tfdA class III gene copy number was approximately 100-fold greater in samples able to mineralize MCPA than in samples able to mineralize 2,4-D, suggesting that tfdA class III gene plays a greater role in the metabolism of MCPA than of 2......Centimetre-scale vertical distribution of mineralization potential was determined for 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) by 96-well microplate radiorespirometric analysis in aquifer sediment...

Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation.

Science.gov (United States)

Ly, Lundi; Chan, Donovan; Aarabi, Mahmoud; Landry, Mylène; Behan, Nathalie A; MacFarlane, Amanda J; Trasler, Jacquetta

2017-07-01

Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation. There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities. Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases. F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA

Relative gene transcription and pathogenicity of enterohemorrhagic Escherichia coli after long-term adaptation to acid and salt stress

DEFF Research Database (Denmark)

Olesen, Inger; Jespersen, Lene

2010-01-01

Relative gene transcription and virulence potential, as measured by a Caco-2 adhesion assay, were investigated for three enterohemorrhagic Escherichia coli (EHEC) strains after long-term adaptation for 24 h to acid (BHI pH 5.5) and salt (BHI 4.5% (w/v) NaCl) stress. Five virulence genes (eae, lpf...... compared to EDL933 (O157:H7, raw hamburger). Long-term adaptation to salt stress significantly increased the adhesion of all three EHEC strains to Caco-2 compared to the non-stressed controls. The present study shows that long-term adaptation to food related stress factors such as acid and salt is capable...... of changing the relative transcription of important virulence and stress response genes and increasing the virulence potential as measured by adhesion to the human colonic epithelial cell line, Caco-2....

Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

Science.gov (United States)

Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2

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Hellberg Michael E

2010-05-01

Full Text Available Abstract Background Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. Results In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus, a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella possess proteins structurally similar to tachylectin-2. Conclusions Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1 part of Oculina's innate immunity repertoire, and 2 evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.

Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene.

Science.gov (United States)

Liu, Xiaofeng; Zhang, Qian; Zhou, Nandi; Tian, Yaping

2017-03-01

Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4-7. And the recoveries of both urease and urethanase activities were more than 50% in 5-25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.

Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1.

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Hattori, T; Terada, T; Hamasuna, S

1995-06-01

Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1.

Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

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Bouchard-Mercier, Annie; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2014-01-01

A large inter-individual variability in the plasma triglyceride (TG) response to an omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs) within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208) participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187) and ACOX1 (rs17583163) genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation. PMID:24647074

Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

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Annie Bouchard-Mercier

2014-03-01

Full Text Available A large inter-individual variability in the plasma triglyceride (TG response to an omega-3 polyunsaturated fatty acid (n-3 PUFA supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208 participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA. Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187 and ACOX1 (rs17583163 genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation.

Effects of tung oilseed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana.

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Chen, Yicun; Cui, Qinqin; Xu, Yongjie; Yang, Susu; Gao, Ming; Wang, Yangdong

2015-08-01

Genetic engineering to produce valuable lipids containing unsaturated fatty acids (UFAs) holds great promise for food and industrial applications. Efforts to genetically modify plants to produce desirable UFAs with single enzymes, however, have had modest success. The key enzymes fatty acid desaturase (FAD) and diacylglycerol acyltransferase (DGAT) are responsible for UFA biosynthesis (a push process) and assembling fatty acids into lipids (a pull process) in plants, respectively. To examine their roles in UFA accumulation, VfFAD2 and VfDGAT2 genes cloned from Vernicia fordii (tung tree) oilseeds were conjugated and transformed into Rhodotorula glutinis and Arabidopsis thaliana via Agrobacterium tumefaciens. Real-time quantitative PCR revealed variable gene expression levels in the transformants, with a much higher level of VfDGAT2 than VfFAD2. The relationship between VfFAD2 expression and linoleic acid (C18:2) increases in R. glutinis (R (2) = 0.98) and A. thaliana (R (2) = 0.857) transformants was statistically linear. The VfDGAT2 expression level was statistically correlated with increased total fatty acid content in R. glutinis (R (2) = 0.962) and A. thaliana (R (2) = 0.8157) transformants. With a similar expression level between single- and two-gene transformants, VfFAD2-VfDGAT2 co-transformants showed a higher linolenic acid (C18:3) yield in R. glutinis (174.36 % increase) and A. thaliana (14.61 % increase), and eicosatrienoic acid (C20:3) was enriched (17.10 % increase) in A. thaliana. Our data suggest that VfFAD2-VfDGAT2 had a synergistic effect on UFA metabolism in R. glutinis, and to a lesser extent, A. thaliana. These results show promise for further genetic engineering of plant lipids to produce desirable UFAs.

TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

Science.gov (United States)

Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

2014-04-01

Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000. Copyright © 2014 Elsevier Ltd. All rights reserved.

The effect of adipocyte and heart fatty acid-binding protein genes on intramuscular fat and backfat content in Meishan crossbred pigs

NARCIS (Netherlands)

Gerbens, F.; Koning, de D.J.; Harders, F.L.; Meuwissen, T.H.E.; Groenen, M.A.M.; Veerkamp, R.L.; Arendonk, van J.A.M.; Pas, te M.F.W.

2000-01-01

Effects of genetic variation in porcine adipocyte and heart fatty acid-binding protein genes, A-FABP and H-FABP, respectively, on intramuscular fat (IMF) content and backfat thickness (BFT) were examined in F2 crossbreds of Meishan and Western pigs. The involvement of each FABP gene in IMF accretion

Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids.

Science.gov (United States)

Olaetxea, Maite; Mora, Verónica; Bacaicoa, Eva; Garnica, María; Fuentes, Marta; Casanova, Esther; Zamarreño, Angel M; Iriarte, Juan C; Etayo, David; Ederra, Iñigo; Gonzalo, Ramón; Baigorri, Roberto; García-Mina, Jose M

2015-12-01

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface. © 2015 American Society of Plant Biologists. All Rights Reserved.

Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells.

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Yenuganti, Vengala Rao; Viergutz, Torsten; Vanselow, Jens

2016-06-01

After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

Science.gov (United States)

Busk, P K; Jensen, A B; Pagès, M

1997-06-01

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

Influence of IL1B, IL6 and IL10 gene variants and plasma fatty acid interaction on metabolic syndrome risk in a cross-sectional population-based study.

Science.gov (United States)

Maintinguer Norde, Marina; Oki, Erica; Ferreira Carioca, Antonio Augusto; Teixeira Damasceno, Nágila Raquel; Fisberg, Regina Mara; Lobo Marchioni, Dirce Maria; Rogero, Marcelo Macedo

2018-04-01

Metabolic syndrome (MetS) is a cluster of interrelated risk factors for type 2 diabetes mellitus, and cardiovascular disease, with underlying inflammatory pathophysiology. Genetic variations and diet are well-known risk factor for MetS, but the interaction between these two factors is less explored. The aim of the study was to evaluate the influence of interaction between SNP of inflammatory genes (encoding interleukin (IL)-6, IL-1β and IL-10) and plasma fatty acids on the odds of MetS, in a population-based cross-sectional study. Among participants of the Health Survey - São Paulo, 301 adults (19-59 y) from whom a blood sample was collected were included. Individuals with and without MetS were compared according to their plasma inflammatory biomarkers, fatty acid profile, and genotype frequency of the IL1B (rs16944, rs1143623, rs1143627, rs1143634 and rs1143643), IL6 (rs1800795, rs1800796 and rs1800797) and IL10 (rs1554286, rs1800871, rs1800872, rs1800890 and rs3024490) genes SNP. The influence of gene-fatty acids interaction on MetS risk was investigated. IL6 gene SNP rs1800795 G allele was associated with higher odds for MetS (OR = 1.88; p = 0.017). Gene-fatty acid interaction was found between the IL1B gene SNP rs116944 and stearic acid (p inter = 0.043), and between rs1143634 and EPA (p inter = 0.017). For the IL10 gene SNP rs1800896, an interaction was found for arachidonic acid (p inter = 0.007) and estimated D5D activity (p inter = 0.019). The IL6 gene SNP rs1800795 G allele is associated with increased odds for MetS. Plasma fatty acid profile interacts with the IL1B and IL10 gene variants to modulate the odds for MetS. This and other interactions of risk factors can account for the unexplained heritability of MetS, and their elucidation can lead to new strategies for genome-customized prevention of MetS. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

Science.gov (United States)

Oberding, Lisa K; Gieg, Lisa M

2018-01-01

Paraffinic n -alkanes (>C 17 ) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n -octacosane (C 28 H 58 ). Compared with that in controls, the consumption of n -octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C 28 H 58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C 28 H 58 -degrading cultures compared with that in controls, suggesting n -octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C 28 H 58 -degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery. IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important

Grape Seed Procyanidins and Cholestyramine Differentially Alter Bile Acid and Cholesterol Homeostatic Gene Expression in Mouse Intestine and Liver.

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Rebecca M Heidker

Full Text Available Bile acid (BA sequestrants, lipid-lowering agents, may be prescribed as a monotherapy or combination therapy to reduce the risk of coronary artery disease. Over 33% of adults in the United States use complementary and alternative medicine strategies, and we recently reported that grape seed procyanidin extract (GSPE reduces enterohepatic BA recirculation as a means to reduce serum triglyceride (TG levels. The current study was therefore designed to assess the effects on BA, cholesterol and TG homeostatic gene expression following co-administration with GSPE and the BA sequestrant, cholestyramine (CHY. Eight-week old male C57BL/6 mice were treated for 4 weeks with either a control or 2% CHY-supplemented diet, after which, they were administered vehicle or GSPE for 14 hours. Liver and intestines were harvested and gene expression was analyzed. BA, cholesterol, non-esterified fatty acid and TG levels were also analyzed in serum and feces. Results reveal that GSPE treatment alone, and co-administration with CHY, regulates BA, cholesterol and TG metabolism differently than CHY administration alone. Notably, GSPE decreased intestinal apical sodium-dependent bile acid transporter (Asbt gene expression, while CHY significantly induced expression. Administration with GSPE or CHY robustly induced hepatic BA biosynthetic gene expression, especially cholesterol 7α-hydroxylase (Cyp7a1, compared to control, while co-administration further enhanced expression. Treatment with CHY induced both intestinal and hepatic cholesterologenic gene expression, while co-administration with GSPE attenuated the CHY-induced increase in the liver but not intestine. CHY also induced hepatic lipogenic gene expression, which was attenuated by co-administration with GSPE. Consequently, a 25% decrease in serum TG levels was observed in the CHY+GSPE group, compared to the CHY group. Collectively, this study presents novel evidence demonstrating that GSPE provides additive and

Relative gene expression of fatty acid synthesis genes at 60 days postpartum in bovine mammary epithelial cells of Surti and Jafarabadi buffaloes

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Mamta Janmeda

2017-05-01

Full Text Available Aim: Aim of the study was to study the relative gene expression of genes associated with fatty acid synthesis at 60 days postpartum (pp in bovine mammary epithelial cells (MECs of Surti and Jafarabadi buffaloes. Materials and Methods: A total of 10 healthy Surti and Jafarabadi buffaloes of each breed were selected at random from Livestock Research Station, Navsari and Cattle Breeding Farm, Junagadh, Gujarat, respectively, for this study. Milk sample was collected from each selected buffalo at day 60 pp from these two breeds to study relative gene expression of major milk fat genes using non-invasive approach of obtaining primary bovine MECs (pBMEC from milk samples. Results: In this study overall, the relative expression of the six major milk lipogenic genes butyrophilin subfamily 1 member A1 (BTN1A1, stearoyl-CoA desaturase (SCD, lipoprotein lipase (LPL, glycerol-3-phosphate acyltransferase mitochondrial (GPAM, acetyl-coenzyme A carboxylase alpha (ACACA, and lipin (LPIN did not show changes in expression patterns at 60th day of lactation in both Surti and Jafarabadi buffaloes. Conclusion: The pBMEC can be successfully recovered from 1500 ml of milk of Surti and Jafarabadi buffaloes using antibody-mediated magnetic bead separation and can be further used for recovering RNA for down step quantification of major milk lipogenic gene expression. The relative expression of the six major milk lipogenic genes BTN1A1, SCD, LPL, GPAM, ACACA, and LPIN did not show changes in expression patterns in both Surti and Jafarabadi buffaloes, suggesting expression levels of lipogenic genes are maintained almost uniform till peak lactation without any significant difference.

Associations of heart and adipocyte fatty acid-binding protein gene expression with intramuscular fat content in pigs

NARCIS (Netherlands)

Gerbens, F.; Verburg, F.J.; Moerkerk, van H.T.; Engel, B.; Buist, W.; Veerkamp, J.H.; Pas, te M.F.

2001-01-01

Intramuscular fat content is a major determinant of meat quality in pigs. Previously, polymorphisms in the adipocyte and heart fatty acid-binding protein genes, A-FABP and H-FABP, have been significantly associated with genetic variation of intramuscular fat content in a Duroc pig population.

Associations of heart and adipocyte fatty acid-binding protein gene expression with intramuscular fat content in pigs.

NARCIS (Netherlands)

Gerbens, F.; Verburg, F.J.; Moerkerk, H.T.B. van; Engel, B.; Buist, W.; Veerkamp, J.H.; Pas, M.F. te

2001-01-01

Intramuscular fat content is a major determinant of meat quality in pigs. Previously, polymorphisms in the adipocyte and heart fatty acid-binding protein genes, A-FABP and H-FABP, have been significantly associated with genetic variation of intramuscular fat content in a Duroc pig population.

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1 -> 3)-glucanase activity at the onset of tobacco defence reactions

DEFF Research Database (Denmark)

Riviere, M.P.; Marais, A.; Ponchet, M.

2008-01-01

silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic...... protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence......The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were...

Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid.

Science.gov (United States)

Yokouchi, Y; Ohsugi, K; Sasaki, H; Kuroiwa, A

1991-10-01

A chicken gene carrying a homeobox highly homologous to the Drosophila muscle segment homeobox (msh) gene was isolated and designated as Msx-1. Conceptual translation from the longest ORF gave a protein of 259 amino acids lacking the conserved hexapeptide. Northern analysis detected a single 2.6 kb transcript. As early as day 2 of incubation, the transcript was detected but was not found in adult tissue. In situ hybridization analysis revealed that Msx-1 expression is closely related to a particular mesenchymal cell lineage during limb bud formation. In early stage embryos, Msx-1 was expressed in the somatopleure. When primordial mesenchyme cells for limb bud were generated from the Wolffian ridge of the somatopleure, Msx-1 expression began to diminish in the posterior half of the limb bud then in the presumptive cartilage-forming mesenchyme. In developing limb buds, remarkable expression was seen in the apical ectodermal ridge (AER), which is responsible for the sustained outgrowth and development of the limb. The Msx-1 transcripts were found in the limb mesenchymal cells in the region covering the necrotic zone and ectodermal cells overlying such mesenchymal cells. Both ectodermal and mesenchymal expression in limb bud were rapidly suppressed by local treatment of retinoic acid which can generate mirror-image duplication of digits. This indicates that retinoic acid alters the marginal presumptive non-cartilage forming mesenchyme cell lineage through suppression of Msx-1 expression.

First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

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Patrick C Y Woo

Full Text Available BACKGROUND: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. METHODOLOGY/PRINCIPAL FINDINGS: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05. There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05. CONCLUSIONS/SIGNIFICANCE: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid

Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus: Implications for the antibacterial mechanism of naringenin.

Science.gov (United States)

Wang, Lang-Hong; Zeng, Xin-An; Wang, Man-Sheng; Brennan, Charles S; Gong, Deming

2018-02-01

In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography-mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20mM. For S. aureus grown with NAR at 0 to 1.47mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC. Copyright © 2017 Elsevier B.V. All rights reserved.

Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPAR{sub β/δ} in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua [Comprehensive Laboratory, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Berggren-Söderlund, Maria; Nilsson-Ehle, Peter [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden); Zhang, Xiaoying, E-mail: zhangxy6689996@163.com [Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Xu, Ning, E-mail: ning.xu@med.lu.se [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden)

2014-02-28

Highlights: • Palmitic acid significantly inhibited APOM gene expression in HepG2 cells. • Palmitic acid could obviously increase PPARB/D mRNA levels in HepG2 cells. • PPAR{sub β/δ} antagonist, GSK3787, had no effect on APOM expression. • GSK3787 could reverse the palmitic acid-induced down-regulation of APOM expression. • Palmitic acid induced suppression of APOM expression is mediated via the PPAR{sub β/δ} pathway. - Abstract: It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPAR{sub β/δ}) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPAR{sub β/δ} pathway.

The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

Science.gov (United States)

Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

2012-11-01

Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

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Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

2015-06-01

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis.

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Ashwell, Melissa S; Ceddia, Ryan P; House, Ralph L; Cassady, Joseph P; Eisen, Eugene J; Eling, Thomas E; Collins, Jennifer B; Grissom, Sherry F; Odle, Jack

2010-09-01

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status. Copyright 2010 Elsevier Inc. All rights reserved.

aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

Science.gov (United States)

de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

1998-01-01

An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

Science.gov (United States)

de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap

1998-01-01

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

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Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2017-02-01

Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7- aminodeacetoxycephalosporanic acid production

DEFF Research Database (Denmark)

Robin, Jarno Jacky Christian; Jakobsen, M.; Beyer, M.

2001-01-01

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this pr......The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase...

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

Science.gov (United States)

Di Renzo, Francesca; Rossi, Federica; Bacchetta, Renato; Prati, Mariangela; Giavini, Erminio; Menegola, Elena

2011-06-01

The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology. © 2011 Wiley-Liss, Inc.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria.

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Bron, Peter A; Benchimol, Marcos G; Lambert, Jolanda; Palumbo, Emmanuelle; Deghorain, Marie; Delcour, Jean; De Vos, Willem M; Kleerebezem, Michiel; Hols, Pascal

2002-11-01

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.

Methylation and silencing of the retinoic acid receptor-β2 gene in cervical cancer

International Nuclear Information System (INIS)

Ivanova, Tatyana; Petrenko, Anatolii; Gritsko, Tatyana; Vinokourova, Svetlana; Eshilev, Ernest; Kobzeva, Vera; Kisseljov, Fjodor; Kisseljova, Natalia

2002-01-01

Expression of the retinoic acid receptor β2 (RAR-β2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-β2 expression remains obscure. We examined whether methylation of RAR-β2 gene could be responsible for this silencing in cervical SCC. Expression of RAR-β2 mRNA and methylation status of the 5' region of RAR-β2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively. In 8 out 20 cervical SCC (40%) the levels of RAR-β2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-β2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-β2 transcript. The RAR-β2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-β2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-β2 gene. These findings suggest that methylation of the 5' region of RAR-β2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation

Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L

DEFF Research Database (Denmark)

Yin, Heng; Kjær, Anders; Fretté, Xavier

2012-01-01

oligosaccharide (COS) and salicylic acid (SA) on both artemisinin production and gene expression related to the biosynthetic pathway of artemisinin. COS up-regulated the transcriptional levels of the genes ADS and TTG1 2.5 fold and 1.8 fold after 48 h individually, whereas SA only up-regulated ADS 2.0 fold after...

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

Science.gov (United States)

Creelman, R A; Tierney, M L; Mullet, J E

1992-06-01

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

Science.gov (United States)

Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

2014-07-01

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Influence of Light and Temperature on Gene Expression Leading to Accumulation of Specific Flavonol Glycosides and Hydroxycinnamic Acid Derivatives in Kale (Brassica oleracea var. sabellica).

Science.gov (United States)

Neugart, Susanne; Krumbein, Angelika; Zrenner, Rita

2016-01-01

Light intensity and temperature are very important signals for the regulation of plant growth and development. Plants subjected to less favorable light or temperature conditions often respond with accumulation of secondary metabolites. Some of these metabolites have been identified as bioactive compounds, considered to exert positive effects on human health when consumed regularly. In order to test a typical range of growth parameters for the winter crop Brassica oleracea var. sabellica, plants were grown either at 400 μmol m(-2) s(-1) or 100 μmol m(-2) s(-1) at 10°C, or at 400 μmol m(-2) s(-1) with 5 or 15°C. The higher light intensity overall increased flavonol content of leaves, favoring the main quercetin glycosides, a caffeic acid monoacylated kaempferol triglycoside, and disinapoyl-gentiobiose. The higher temperature mainly increased the hydroxycinnamic acid derivative disinapoyl-gentiobiose, while at lower temperature synthesis is in favor of very complex sinapic acid acylated flavonol tetraglycosides such as kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside. A global analysis of light and temperature dependent alterations of gene expression in B. oleracea var. sabellica leaves was performed with the most comprehensive Brassica microarray. When compared to the light experiment much less genes were differentially expressed in kale leaves grown at 5 or 15°C. A structured evaluation of differentially expressed genes revealed the expected enrichment in the functional categories of e.g. protein degradation at different light intensities or phytohormone metabolism at different temperature. Genes of the secondary metabolism namely phenylpropanoids are significantly enriched with both treatments. Thus, the genome of B. oleracea was screened for predicted genes putatively involved in the biosynthesis of flavonoids and hydroxycinnamic acid derivatives. All identified B. oleracea genes were analyzed for their most specific 60-mer oligonucleotides present on the

Molecular cloning and expression of the hyu genes from Microbacterium liquefaciens AJ 3912, responsible for the conversion of 5-substituted hydantoins to alpha-amino acids, in Escherichia coli.

Science.gov (United States)

Suzuki, Shun'ichi; Takenaka, Yasuhiro; Onishi, Norimasa; Yokozeki, Kenzo

2005-08-01

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi.

Science.gov (United States)

Zhang, De-Huai; Jiang, Lu-Xi; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2017-06-14

The squalene epoxidase (SE) gene from the biosynthetic pathway of ganoderic acid (GA) was cloned and overexpressed in Ganoderma lingzhi. The strain that overexpressed the SE produced approximately 2 times more GA molecules than the wild-type (WT) strain. Moreover, SE overexpression upregulated lanosterol synthase gene expression in the biosynthetic pathway. These results indicated that SE stimulates GA accumulation. Then, the SE and 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) genes were simultaneously overexpressed in G. lingzhi. Compared with the individual overexpression of SE or HMGR, the combined overexpression of the two genes further enhanced individual GA production. The overexpressing strain produced maximum GA-T, GA-S, GA-Mk, and GA-Me contents of 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/100 mg dry weight, respectively. These values were 5.9, 4.5, 2.4, and 5.8 times higher than those produced by the WT strain. This is the first example of the successful manipulation of multiple biosynthetic genes to improve GA content in G. lingzhi.

Effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine

Directory of Open Access Journals (Sweden)

Ebrahim Amini

2016-04-01

Full Text Available Background: Linoleic acid (LA is a polyunsaturated fatty acid present in high concentrations in follicular fluid, when added to maturation culture media, it affects oocyte competence. Objective: In the present study, we investigated effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine Materials and Methods: The experiments conducted on 450 ovine Cumulus-oocyte complexes (COCs with homogenous ooplasm and more than two compact layers of cumulus cells. For in vitro maturation COCs were randomly allocated into four treatment groups for 24 hr period. Treatment groups were as follow: control maturation media, 0 μM LA, 50 μM LA, 100 μM LA and 200 μM LA. The cumulus cell expansion and blastocysts rates were recorded. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to apoptotic gene expression by real-time PCR. Results: Highest concentration (200 μM/mL of LA significantly decreased the rate of fully expanded cumulus cells 24 hr after in vitro maturation (IVM and the percentage of blastocyste rate compared with the control (p<0.05. These inhibitory effects were associated with an increased in relative mRNA expression of Bax (Bcl-2- associated X gene compared with controls. Conclusion: Data obtained in present study suggest that low concentration of LA used for maturation had no deleterious effect on subsequent embryonic development compared to high concentration of LA. Relative expression of Bcl-2 (B-cell lymphoma 2 and Bax in embryos seems to be associated with LA concentration.

Newborn serum retinoic acid level is associated with variants of genes in the retinol metabolism pathway.

Science.gov (United States)

Manolescu, Daniel C; El-Kares, Reyhan; Lakhal-Chaieb, Lajmi; Montpetit, Alexandre; Bhat, Pangala V; Goodyer, Paul

2010-06-01

Retinoic acid (RA) is a critical regulator of gene expression during embryonic development. In rodents, moderate maternal vitamin A deficiency leads to subtle morphogenetic defects and inactivation of RA pathway genes causes major disturbances of embryogenesis. In this study, we quantified RA in umbilical cord blood of 145 healthy full-term Caucasian infants from Montreal. Sixty seven percent of values were ROL). However, we found that the (A) allele of the rs12591551 single nucleotide polymorphism (SNP) in the ALDH1A2 gene (ALDH1A2rs12591551(A)), occurring in 19% of newborns, was associated with 2.5-fold higher serum RA levels. ALDH1A2 encodes retinaldehyde dehydrogenase (RALDH) 2, which synthesizes RA in fetal tissues. We also found that homozygosity for the (A) allele of the rs12724719 SNP in the CRABP2 gene (CRABP2rs12724719(A/A)) was associated with 4.4-fold increase in umbilical cord serum RA. CRABP2 facilitates RA binding to its cognate receptor complex and transfer to the nucleus. We hypothesize that individual variation in RA pathway genes may account for subtle variations in RA-dependent human embryogenesis.

New gene cluster from the thermophile Bacillus fordii MH602 in the conversion of DL-5-substituted hydantoins to L-amino acids.

Science.gov (United States)

Mei, Yan-Zhen; Wan, Yong-Min; He, Bing-Fang; Ying, Han-Jie; Ouyang, Ping-Kai

2009-12-01

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.

Heme oxygenase-1 and abscisic acid effects MAPK´s gene expression in soybean seeds

International Nuclear Information System (INIS)

Giacometti, R.; Santa Cruz, D.; Noriega, G.; Balestrasse, K.

2012-01-01

In soybean previous studies enabled the identification of MAPK3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. In this study the effects of different compounds related to hemeoxygenase (HO-1) biosynthesis on mitogen-activated protein kinase (MAPK’s) genes expression in soybean seeds were tested. To this end, 20μM hemine, 22μM ZnPPIX, 0.5mM furidine or 100μM 8-bromoguanosine 3',5'-cyclic monophosphate (8Br) were added to pre-hydrated seeds for 5 days. MAPK’s genes expression was enhanced in seeds treated with hemine. This result indicates that heme catabolism could be involved in the signaling mediated by this cascade pathway. To confirm this hypothesis experiments were carried out in the precsence of ZnPPIX, a potent irreversible HO-1 inhibitor. In this case, no gene induction was observed. On the other hand, 8Br, a cGMP analog, induced HO-1 gene expression but did not modulate MAPK’s, indicating that this effect could not be mediated by cGMP. When the action of furidine, an abscisic acid inhibitor, was tested a diminution of HO-1 gene expression was observed. In this regard, MAPK’s showed a different response, being MAPK6 the only transcript that showed a diminished respect to controls, while MAPK3 mRNA as well as MAPKK1 was enhanced. These results were confirmed by western blotting and activity determinations. (authors)

Identification of Genes in Saccharomyces cerevisiae that Are Haploinsufficient for Overcoming Amino Acid Starvation

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Nancy S. Bae

2017-04-01

Full Text Available The yeast Saccharomyces cerevisiae responds to amino acid deprivation by activating a pathway conserved in eukaryotes to overcome the starvation stress. We have screened the entire yeast heterozygous deletion collection to identify strains haploinsufficient for growth in the presence of sulfometuron methyl, which causes starvation for isoleucine and valine. We have discovered that cells devoid of MET15 are sensitive to sulfometuron methyl, and loss of heterozygosity at the MET15 locus can complicate screening the heterozygous deletion collection. We identified 138 cases of loss of heterozygosity in this screen. After eliminating the issues of the MET15 loss of heterozygosity, strains isolated from the collection were retested on sulfometuron methyl. To determine the general effect of the mutations for a starvation response, SMM-sensitive strains were tested for the ability to grow in the presence of canavanine, which induces arginine starvation, and strains that were MET15 were also tested for growth in the presence of ethionine, which causes methionine starvation. Many of the genes identified in our study were not previously identified as starvation-responsive genes, including a number of essential genes that are not easily screened in a systematic way. The genes identified span a broad range of biological functions, including many involved in some level of gene expression. Several unnamed proteins have also been identified, giving a clue as to possible functions of the encoded proteins.

Comparison of Fatty Acid and Gene Profiles in Skeletal Muscle in Normal and Obese C57BL/6J Mice before and after Blunt Muscle Injury

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Jens-Uwe Werner

2018-01-01

Full Text Available Injury and obesity are two major health burdens affecting millions of people worldwide. Obesity is recognized as a state of chronic inflammation accompanied by various co-morbidities like T2D or cardiovascular diseases. There is increasing evidence that obesity impairs muscle regeneration, which is mainly due to chronic inflammation and to excessive accumulation of lipids in adipose and non-adipose tissue. To compare fatty acid profiles and changes in gene expression at different time points after muscle injury, we used an established drop tower-based model with a defined force input to damage the extensor iliotibialis anticus on the left hind limb of female C57BL/6J mice of normal weight and obese mice. Although most changes in fatty acid content in muscle tissue are diet related, levels of eicosaenoic (normal weight and DHG-linolenic acid (obese in the phospholipid and docosahexaenoic acid (normal weight in the triglyceride fraction are altered after injury. Furthermore, changes in gene transcription were detected in 3829 genes in muscles of normal weight mice, whereas only 287 genes were altered in muscles of obese mice after trauma. Alterations were found within several pathways, among them notch-signaling, insulin-signaling, sonic hedgehog-signaling, apoptosis related pathways, fat metabolism related cholesterol homeostasis, fatty acid biosynthetic process, fatty acid elongation, and acyl-CoA metabolic process. We could show that genes involved in fat metabolism are affected 3 days after trauma induction mostly in normal weight but not in obese mice. The strongest effects were observed in normal weight mice for Alox5ap, the activating protein for leukotriene synthesis, and Apobec1, an enzyme substantial for LDL synthesis. In summary, we show that obesity changes the fat content of skeletal muscle and generally shows a negative impact upon blunt muscle injury on various cellular processes, among them fatty acid related metabolism, notch

Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

International Nuclear Information System (INIS)

Walters, M.W.; Bjork, J.A.; Wallace, K.B.

2009-01-01

Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague-Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3-4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

Modulation of LXR-α and the effector genes by Ascorbic acid and Statins in psoriatic keratinocytes.

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Soodgupta, Deepti; Kaul, Deepak; Kanwar, A J; Parsad, Davinder

2014-12-01

Recent studies have revealed critical roles that nuclear receptors like LXR-α (Liver X Receptor- alpha) plays as a class of post-transcriptional gene regulator in skin development and diseases. Keeping in view the fact that LXR-α plays crucial role in keratinocyte proliferation and differentiation, it becomes imperative to dissect the pathways and role of LXR-α genomics in the pathogenesis of psoriasis with ultimate aim to explore novel preventive/therapeutic strategies as treatment options. To explore the effects of agonists and activators of LXR-α on its own gene expression and the putative targets in psoriatic keratinocytes. Identification of promoter sequences for (vitamin D receptor) VDR and Catalase were done using in silico analysis followed by β-galactosidase (β-gal) reporter plasmid assay in keratinocytes from clinically heathy subjects. Determination of relative levels of LXR-α,VDR and catalase in control versus treated cells upon activation of LXR-α with Atorvastatin + 22R hydroxycholestrol and Ascorbic acid + 22R hydroxycholestrol was done by PCR and Cell Proliferation Assay. The cells transfected with the reporter plasmid element for VDR and catalase showed more than 5 and 4 fold increase respectively in the β-gal activity compared to the control. An increase of 55% in LXR-α gene expression at RNA level was observed in Atorvastatin + 22-R hydroxycholestrol compared to 24% in Ascorbic acid + 22-ROH cholesterol. The expression of the VDR and Catalase was significantly increased in both treated keratinocytes compared to its normal counterpart.

Facile preparation of a cationic poly(amino acid) vesicle for potential drug and gene co-delivery

Science.gov (United States)

Ding, Jianxun; Xiao, Chunsheng; He, Chaoliang; Li, Mingqiang; Li, Di; Zhuang, Xiuli; Chen, Xuesi

2011-12-01

A novel pH-responsive poly(amino acid) grafted with oligocation was prepared through the combination of ring-opening polymerization (ROP) and subsequent atom transfer radical polymerization (ATRP). Firstly, poly(γ-2-chloroethyl-L-glutamate) (PCELG) with a pendent 2-chloroethyl group was synthesized through ROP of γ-2-chloroethyl-L-glutamate N-carboxyanhydride (CELG NCA) using n-hexylamine as the initiator. Then, PCELG was used to initiate the ARTP of 2-aminoethyl methacrylate hydrochloride (AMA), yielding poly(L-glutamate)-graft-oligo(2-aminoethyl methacrylate hydrochloride) (PLG-g-OAMA). The pKa of PLG-g-OAMA was 7.3 established by the acid-base titration method. The amphiphilic poly(amino acid) could directly self-assemble into a vesicle in PBS. The vesicle was characterized by TEM and DLS. Hydrophilic DOX·HCl was loaded into the hollow core of the vesicle. The in vitro release behavior of DOX·HCl from the vesicle in PBS could be adjusted by the solution pH. In vitro cell experiments revealed that the vesicle could reduce the toxicity of the DOX·HCl. In addition, the preliminary gel retardation assay displayed that PLG-g-OAMA could efficiently bind DNA at a PLG-g-OAMA/DNA weight ratio of 0.3 or above, indicating its potential use as a gene carrier. More in-depth studies of the PLG-g-OAMA vesicle for drug and gene co-delivery in vitro and in vivo are in progress.

Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

DEFF Research Database (Denmark)

Maestrini, E.; Lai, C.; Marlow, A.

1999-01-01

Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study on...

The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate–Dependent Nucleic Acid Demethylase

Science.gov (United States)

Gerken, Thomas; Girard, Christophe A.; Tung, Yi-Chun Loraine; Webby, Celia J.; Saudek, Vladimir; Hewitson, Kirsty S.; Yeo, Giles S. H.; McDonough, Michael A.; Cunliffe, Sharon; McNeill, Luke A.; Galvanovskis, Juris; Rorsman, Patrik; Robins, Peter; Prieur, Xavier; Coll, Anthony P.; Ma, Marcella; Jovanovic, Zorica; Farooqi, I. Sadaf; Sedgwick, Barbara; Barroso, Inês; Lindahl, Tomas; Ponting, Chris P.; Ashcroft, Frances M.; O'Rahilly, Stephen; Schofield, Christopher J.

2009-01-01

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate–dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass. PMID:17991826

Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

Science.gov (United States)

Wiese, A; Syldatk, C; Mattes, R; Altenbuchner, J

2001-09-01

Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

Impact of Branched-Chain Amino Acid Catabolism on Fatty Acid and Alkene Biosynthesis in Micrococcus luteus.

Science.gov (United States)

Surger, Maximilian J; Angelov, Angel; Stier, Philipp; Übelacker, Maria; Liebl, Wolfgang

2018-01-01

Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus , whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain α-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus . We also show how

Identification and characterization of polymorphisms at the HSA a1-acid glycoprotein (ORM* gene locus in Caucasians

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Owczarek Catherine M.

2002-01-01

Full Text Available Human alpha1-acid glycoprotein (AGP or orosomucoid (ORM is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a panel of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Non-random associations were found between the BamHI, EcoRI, BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the indiviuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms will prove useful for other population and forensic studies.

Lysophosphatidic acid induces expression of genes in human oral keratinocytes involved in wound healing.

Science.gov (United States)

Thorlakson, Hong Huynh; Engen, Stian Andre; Schreurs, Olav; Schenck, Karl; Blix, Inger Johanne Schytte

2017-08-01

Epithelial cells participate in wound healing by covering wounds, but also as important mediators of wound healing processes. Topical application of the phospholipid growth factor lysophosphatidic acid (LPA) accelerates dermal wound healing and we hypothesized that LPA can play a role in human oral wound healing through its effects on human oral keratinocytes (HOK). HOK were isolated from gingival biopsies and exposed to LPA. The LPA receptor profile, signal transduction pathways, gene expression and secretion of selected cytokines were analyzed. HOK expressed the receptors LPA 1 , LPA 5 and LPA 6 and LPA activated the ERK1/2, JNK and p38 intracellular pathways, substantiated by secretion of IL-6 and IL-8. The early (2h) and intermediate (6h) gene expression profiles of HOK after LPA treatment showed a wide array of regulated genes. The majority of the strongest upregulated genes were related to chemotaxis and inflammation, and became downregulated after 6h. At 6h, genes coding for factors involved in extracellular matrix remodeling and re-epithelialization became highly expressed. IL-36γ, not earlier known to be regulated by LPA, was strongly transcribed and translated but not secreted. After stimulation with LPA, HOK responded by regulating factors and genes that are essential in wound healing processes. As LPA is found in saliva and is released by activated cells after wounding, our results indicate that LPA has a favorable physiological role in oral wound healing. This may further point towards a beneficial role for application of LPA on oral surgical or chronic wounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and NMR characterization of dendrimers based on 2, 2-bis-(hydroxymethyl-propanoic acid (bis-HMPA containing peripheral amino acid residues for gene transfection

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Silvana Alfei

2017-08-01

Full Text Available Dendrimers, the emerging man made, highly branched, star-shaped macromolecules with nanometer-scale dimensions are well known for their well defined and high controlled architecture, their versatility and high functionality and are of eminent interest in nanomedical applications such as drug delivery, gene transfection, and imaging. In this paper, versatile protocols for the synthesis of polyester-based, hydrolysable, polycationic dendrimers have been setup. A fourth generation dendrimer equipped with 48 peripheral hydroxyl groups was prepared from 2,2-bis(hydroxymethylpropanoic acid and was used for grafting BOC-amino acids or as “hypercores” on which dendrons functionalized with BOC-amino acids were attached. A library of 15 polycationic homo- and hetero-dendrimers in the form of hydrochloride was obtained. Their structures and composition were confirmed by NMR analysis and by experimental molecular weight computed by volumetric titration. Their buffer capacity and results obtained from cytotoxicity assays and tests of binding with both pDNA and siRNA were very satisfactory.

Fatty acid desaturase (FADS gene polymorphisms and insulin resistance in association with serum phospholipid polyunsaturated fatty acid composition in healthy Korean men: cross-sectional study

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Yang Long In

2011-04-01

Full Text Available Abstract Background We investigated the relationship between fatty acid desaturase (FADS gene polymorphisms and insulin resistance (IR in association with serum phospholipid polyunsaturated fatty acid (FA composition in healthy Korean men. Methods Healthy men (n = 576, 30 ~ 79 years old were genotyped for rs174537 near FADS1 (FEN1-10154G>T, FADS2 (rs174575C>G, rs2727270C>T, and FADS3 (rs1000778C>T SNPs. Dietary intake, serum phospholipid FA composition and HOMA-IR were measured. Results Fasting insulin and HOMA-IR were significantly higher in the rs174575G allele carriers than the CC homozygotes, but lower in the rs2727270T allele carriers than the CC homozygotes. The proportion of linoleic acid (18:2ω-6, LA was higher in the minor allele carriers of FEN1-10154G>T, rs174575C>G and rs2727270C>T than the major homozygotes, respectively. On the other hand, the proportions of dihomo-γ-linolenic acid (20:3ω-6, DGLA and arachidonic acid (20:4ω-6, AA in serum phospholipids were significantly lower in the minor allele carriers of FEN1-10154 G>T carriers and rs2727270C>T than the major homozygotes respectively. AA was also significantly lower in the rs1000778T allele carriers than the CC homozygotes. HOMA-IR positively correlated with LA and DGLA and negatively with AA/DGLA in total subjects. Interestingly, rs174575G allele carriers showed remarkably higher HOMA-IR than the CC homozygotes when subjects had higher proportions of DLGA (≥1.412% in total serum phospholipid FA composition (P for interaction = 0.009 or of AA (≥4.573% (P for interaction = 0.047. Conclusion HOMA-IR is associated with FADS gene cluster as well as with FA composition in serum phospholipids. Additionally, HOMA-IR may be modulated by the interaction between rs174575C>G and the proportion of DGLA or AA in serum phospholipids.

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

Science.gov (United States)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease

Science.gov (United States)

Sherif , S. M.; Shukla, M. R.; Murch, S. J.; Bernier, L.; Saxena, P. K.

2016-01-01

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions. PMID:26902398

Can polymorphisms in the fatty acid desaturase (FADS) gene cluster alter the effects of fish oil supplementation on plasma and erythrocyte fatty acid profiles? An exploratory study.

Science.gov (United States)

Meldrum, Suzanne J; Li, Yuchun; Zhang, Guicheng; Heaton, Alexandra E M; D'Vaz, Nina; Manz, Judith; Reischl, Eva; Koletzko, Berthold V; Prescott, Susan L; Simmer, Karen

2017-09-19

The enzymes encoded by fatty acid desaturases (FADS) genes determine the desaturation of long-chain polyunsaturated fatty acids (LCPUFA). We investigated if haplotype and single nucleotide polymorphisms (SNPs) in FADS gene cluster can influence LCPUFA status in infants who received either fish oil or placebo supplementation. Children enrolled in the Infant Fish Oil Supplementation Study (IFOS) were randomly allocated to receive either fish oil or placebo from birth to 6 months of age. Blood was collected at 6 months of age for the measurement of fatty acids and for DNA extraction. A total of 276 participant DNA samples underwent genotyping, and 126 erythrocyte and 133 plasma fatty acid measurements were available for analysis. Twenty-two FADS SNPs were selected on the basis of literature and linkage disequilibrium patterns identified from the HapMap data. Haplotype construction was completed using PHASE. For participants allocated to the fish oil group who had two copies of the FADS1 haplotype consisting of SNP minor alleles, DHA levels were significantly higher compared to other haplotypes. This finding was not observed for the placebo group. Furthermore, for members of the fish oil group only, the minor homozygous carriers of all the FADS1 SNPs investigated had significantly higher DHA than other genotypes (rs174545, rs174546, rs174548, rs174553, rs174556, rs174537, rs174448, and rs174455). Overall results of this preliminary study suggest that supplementation with fish oil may only significantly increase DHA in minor allele carriers of FADS1 SNPs. Further research is required to confirm this novel finding.

Mms Sensitivity of All Amino Acid-Requiring Mutants in Aspergillus and Its Suppression by Mutations in a Single Gene

OpenAIRE

Käfer, Etta

1987-01-01

All available amino acid-requiring mutants of Aspergillus nidulans were found to be hypersensitive to MMS (methyl methanesulfonate) to various degrees. On MMS media, secondary mutations could be selected which suppress this MMS sensitivity but do not affect the requirement. Many such mutations were analyzed and found to be alleles of one gene, smsA (= suppressor of MMS sensitivity), which mapped distal on the right arm of chromosome V. This gene is more likely to be involved in general regula...

Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

NARCIS (Netherlands)

Mach Casellas, N.; Jacobs, A.A.A.; Kruijt, L.; Baal, van J.; Smits, M.A.

2011-01-01

The aim of this study was to determine the effects of supplementing unprotected dietary unsaturated fatty acids (UFAs) from different plant oils on gene expression in the mammary gland of grazing dairy cows. A total of 28 Holstein–Friesian dairy cows in mid-lactation were blocked according to

Quantitative Determination of Ceramide Molecular Species in Dendritic Cells

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Samar Al Makdessi

2016-09-01

Full Text Available Background/Aims: The activation of acid sphingomyelinase by cellular stress or receptors or the de novo synthesis lead to the formation of ceramide (N-acylsphingosine, which in turn modifies the biophysical properties of cellular membrane and greatly amplifies the intensity of the initial signal. Ceramide, which acts by re-organizing a given signalosome rather than being a second messenger, has many functions in infection biology, cancer, cardiovascular syndromes, and immune regulation. Experimental studies on the infection of human cells with different bacterial agents demonstrated the activation of the acid sphingomyelinase/ceramide system. Moreover, the release of ceramide was found to be a requisite for the uptake of the pathogen. Considering the particular importance of the cellular role of ceramide, it was necessary to develop sensitive and accurate methods for its quantification. Methods: Here, we describe a method quantifying ceramide in dendritic cells and defining the different fatty acids (FA bound to sphingosine. The main steps of the method include extraction of total lipids, separation of the ceramide by thin-layer chromatography, derivatization of ceramide-fatty acids (Cer-FA, and quantitation of these acids in their methyl form by gas chromatography on polar capillary columns. The identification of FA was achieved by means of known standards and confirmed by mass spectrometry. Results: FA ranging between C10 and C24 could be detected and quantified. The concentration of the sum of Cer-FA amounted to 14.88 ± 8.98 nmol/106 cells (n=10. Oleic acid, which accounted for approximately half of Cer-FA (7.73 ± 6.52 nmol/106 cells was the predominant fatty acid followed by palmitic acid (3.47 ± 1.54 nmol/106 cells. Conclusion: This highly sensitive method allows the quantification of different molecular species of ceramides.

Genetic variation in toll-like receptors and retinoic acid-inducible gene I and outcome of hepatitis C virus infection

DEFF Research Database (Denmark)

Clausen, L N; Ladelund, S; Weis, N

2014-01-01

We evaluated the effects of genetic variation in toll-like receptors (TLR), retinoic acid-inducible gene I (RIG-I) and their signalling pathways on spontaneous hepatitis C virus (HCV) resolution. We screened 95 single-nucleotide polymorphisms (SNPs) in 22 genes. SNPs significantly associated...... with resolution in the discovery cohort were genotyped in a validation cohort. Multivariate logistic regression adjusted for sex, hepatitis B surface antigen, HIV infection and the interleukin-28B rs12979860 SNP was performed in the combined cohort. Haplotype reconstruction and linkage disequilibrium analysis...

Enhanced apoptosis and radiosensitization by combined 13-CIS-retinoic acid and interferon-α2a; role of RAR-β gene

International Nuclear Information System (INIS)

Ryu, Samuel; Stein, Joseph P.; Chung, Chung T.; Lee, Yong J.; Kim, Jae Ho

2001-01-01

Purpose: Combined use of 13-cis-retinoic acid (cRA) and interferon-α2a (IFNα) induced significant radiosensitization in human cervical cancer ME-180 cell line, whereas it failed to achieve similar radiation enhancement in HeLa cells. The differential radiosensitization could be from the difference of retinoic acid receptor (RAR) expression because RAR-β was highly expressed in ME-180 cells in contrast to the HeLa cells where RAR-β was not detectable. We examined the role of this gene in mediating radiosensitization by cRA and IFNα, and explored the mechanism of radiation-induced cell killing. Methods and Materials: Human cervical cancer cell lines, ME-180 and HeLa, were treated with cRA and IFNα followed by radiation. Apoptosis and radiosensitization were quantitated by TUNEL assay (in situ DNA nick end labeling) and colony-forming ability of surviving cells. The cells were transfected with bcl-2 gene and RAR-β gene to test the role of these genes in mediating radiosensitization and apoptosis. Results: Synergistic radiosensitization and apoptosis was observed by combined use of cRA and IFNα with radiation in ME-180 cells which express high level of RAR-β mRNA, whereas these were not seen in HeLa cells where RAR-β mRNA is not detectable. Both radiosensitization and apoptosis were abolished by bcl-2 gene in ME-180 cells. RAR-β gene transfection induced similar radiation enhancement and apoptosis in HeLa cells. Conclusion: Apoptosis and radiation response were enhanced in the cells with high level of RAR-β mRNA expression. The RAR-β gene appears to mediate the radiation-induced apoptosis by cRA and IFNα. These findings indicate that presence of RAR-β in the cancer cells could be exploited for patient selection in using these drugs for apoptosis and radiosensitization

A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple.

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Bai, Yang; Dougherty, Laura; Li, Mingjun; Fazio, Gennaro; Cheng, Lailiang; Xu, Kenong

2012-08-01

Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R (2) = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS(1455) that targets the SNP(1455) in Ma1 showed that the CAPS(1455A) allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma

Coordination of gene expression of arachidonic and docosahexaenoic acid cascade enzymes during human brain development and aging.

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Ryan, Veronica H; Primiani, Christopher T; Rao, Jagadeesh S; Ahn, Kwangmi; Rapoport, Stanley I; Blanchard, Helene

2014-01-01

The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA) participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades. AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging. The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism. Expression patterns were split into Development (0 to 20 years) and Aging (21 to 78 years) intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2), cyclooxygenases (COX)-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA) and PTGS2 (COX-2) genes at 1q25, highly inter-correlated genes were at distant chromosomal loci. Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease.

Coordination of gene expression of arachidonic and docosahexaenoic acid cascade enzymes during human brain development and aging.

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Veronica H Ryan

Full Text Available The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades.AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging.The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism.Expression patterns were split into Development (0 to 20 years and Aging (21 to 78 years intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2, cyclooxygenases (COX-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA and PTGS2 (COX-2 genes at 1q25, highly inter-correlated genes were at distant chromosomal loci.Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease.

Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.).

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Onishi, M; Tachi, H; Kojima, T; Shiraiwa, M; Takahara, H

2006-10-01

We identified a novel salt-inducible soybean gene encoding an acidic-isoform of pathogenesis-related protein group 5 (PR-5 protein). The soybean PR-5-homologous gene, designated as Glycine max osmotin-like protein, acidic isoform (GmOLPa)), encodes a putative polypeptide having an N-terminal signal peptide. The mature GmOLPa protein without the signal peptide has a calculated molecular mass of 21.5 kDa and a pI value of 4.4, and was distinguishable from a known PR-5-homologous gene of soybean (namely P21 protein) through examination of the structural features. A comparison with two intracellular salt-inducible PR-5 proteins, tobacco osmotin and tomato NP24, revealed that GmOLPa did not have a C-terminal extension sequence functioning as a vacuole-targeting motif. The GmOLPa gene was transcribed constitutively in the soybean root and was induced almost exclusively in the root during 24 h of high-salt stress (300 mM NaCl). Interestingly, GmOLPa gene expression in the stem and leaf, not observed until 24 h, was markedly induced at 48 and 72 h after commencement of the high-salt stress. Abscisic acid (ABA) and dehydration also induced expression of the GmOLPa gene in the root; additionally, dehydration slightly induced expression in the stem and leaf. In fact, the 5'-upstream sequence of the GmOLPa gene contained several putative cis-elements known to be involved in responsiveness to ABA and dehydration, e.g. ABA-responsive element (ABRE), MYB/MYC, and low temperature-responsive element (LTRE). These results suggested that GmOLPa may function as a protective PR-5 protein in the extracellular space of the soybean root in response to high-salt stress and dehydration.

Association between Polymorphisms in the Fatty Acid Desaturase Gene Cluster and the Plasma Triacylglycerol Response to an n-3 PUFA Supplementation

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Cormier, Hubert; Rudkowska, Iwona; Paradis, Ann-Marie; Thifault, Elisabeth; Garneau, Véronique; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2012-01-01

Eicosapentaenoic and docosahexaenoic acids have been reported to have a variety of beneficial effects on cardiovascular disease risk factors. However, a large inter-individual variability in the plasma lipid response to an omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation is observed in different studies. Genetic variations may influence plasma lipid responsiveness. The aim of the present study was to examine the effects of a supplementation with n-3 PUFA on the plasma lipid profile in relation to the presence of single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster. A total of 208 subjects from Quebec City area were supplemented with 3 g/day of n-3 PUFA, during six weeks. In a statistical model including the effect of the genotype, the supplementation and the genotype by supplementation interaction, SNP rs174546 was significantly associated (p = 0.02) with plasma triglyceride (TG) levels, pre- and post-supplementation. The n-3 supplementation had an independent effect on plasma TG levels and no significant genotype by supplementation interaction effects were observed. In summary, our data support the notion that the FADS gene cluster is a major determinant of plasma TG levels. SNP rs174546 may be an important SNP associated with plasma TG levels and FADS1 gene expression independently of a nutritional intervention with n-3 PUFA. PMID:23016130

Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs.

Science.gov (United States)

Xu, Ming; Zhang, Qiufang; Li, Pin-Lan; Nguyen, Thaison; Li, Xiang; Zhang, Yang

2016-01-01

Acid sphingomyelinase (ASM; gene symbol Smpd1) has been shown to play a crucial role in autophagy maturation by controlling lysosomal fusion with autophagosomes in coronary arterial smooth muscle cells (CASMCs). However, the underlying molecular mechanism by which ASM controls autophagolysosomal fusion remains unknown. In primary cultured CASMCs, lysosomal Ca2+ induced by 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer) was markedly attenuated by ASM deficiency or TRPML1 gene silencing suggesting that ASM signaling is required for TRPML1 channel activity and subsequent lysosomal Ca(2+) release. In these CASMCs, ASM deficiency or TRPML1 gene silencing markedly inhibited 7-Ket-induced dynein activation. In addition, 7-Ket-induced autophagosome trafficking, an event associated with lysosomal Ca(2+) release and dynein activity, was significantly inhibited in ASM-deficient (Smpd1(-/-)) CASMCs compared to that in Smpd1(+/+) CASMCs. Finally, overexpression of TRPML1 proteins restored 7-Ket-induced lysosomal Ca(2+) release and autophagosome trafficking in Smpd1-/- CASMCs. Collectively, these results suggest that ASM plays a critical role in regulating lysosomal TRPML1-Ca(2+) signaling and subsequent dynein-mediated autophagosome trafficking, which leads its role in controlling autophagy maturation in CASMCs under atherogenic stimulation.

Transcriptome analysis of H2O2-treated wheat seedlings reveals a H2O2-responsive fatty acid desaturase gene participating in powdery mildew resistance.

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Aili Li

Full Text Available Hydrogen peroxide (H(2O(2 plays important roles in plant biotic and abiotic stress responses. However, the effect of H(2O(2 stress on the bread wheat transcriptome is still lacking. To investigate the cellular and metabolic responses triggered by H(2O(2, we performed an mRNA tag analysis of wheat seedlings under 10 mM H(2O(2 treatment for 6 hour in one powdery mildew (PM resistant (PmA and two susceptible (Cha and Han lines. In total, 6,156, 6,875 and 3,276 transcripts were found to be differentially expressed in PmA, Han and Cha respectively. Among them, 260 genes exhibited consistent expression patterns in all three wheat lines and may represent a subset of basal H(2O(2 responsive genes that were associated with cell defense, signal transduction, photosynthesis, carbohydrate metabolism, lipid metabolism, redox homeostasis, and transport. Among genes specific to PmA, 'transport' activity was significantly enriched in Gene Ontology analysis. MapMan classification showed that, while both up- and down- regulations were observed for auxin, abscisic acid, and brassinolides signaling genes, the jasmonic acid and ethylene signaling pathway genes were all up-regulated, suggesting H(2O(2-enhanced JA/Et functions in PmA. To further study whether any of these genes were involved in wheat PM response, 19 H(2O(2-responsive putative defense related genes were assayed in wheat seedlings infected with Blumeria graminis f. sp. tritici (Bgt. Eight of these genes were found to be co-regulated by H(2O(2 and Bgt, among which a fatty acid desaturase gene TaFAD was then confirmed by virus induced gene silencing (VIGS to be required for the PM resistance. Together, our data presents the first global picture of the wheat transcriptome under H(2O(2 stress and uncovers potential links between H(2O(2 and Bgt responses, hence providing important candidate genes for the PM resistance in wheat.

Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

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Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

2011-09-01

A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya

NARCIS (Netherlands)

Malik, Amarila; Radji, Maksum; Kralj, Slavko; Dijkhuizen, Lubbert

2009-01-01

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and

Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid-responsive genes.

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Hermans, Christian; Vuylsteke, Marnik; Coppens, Frederik; Craciun, Adrian; Inzé, Dirk; Verbruggen, Nathalie

2010-07-01

*Plant growth and development ultimately depend on environmental variables such as the availability of essential minerals. Unravelling how nutrients affect gene expression will help to understand how they regulate plant growth. *This study reports the early transcriptomic response to magnesium (Mg) deprivation in Arabidopsis. Whole-genome transcriptome was studied in the roots and young mature leaves 4, 8 and 28 h after the removal of Mg from the nutrient solution. *The highest number of regulated genes was first observed in the roots. Contrary to other mineral deficiencies, Mg depletion did not induce a higher expression of annotated genes in Mg uptake. Remarkable responses include the perturbation of the central oscillator of the circadian clock in roots and the triggering of abscisic acid (ABA) signalling, with half of the up-regulated Mg genes in leaves being ABA-responsive. However, no change in ABA content was observed. *The specificity of the response of some Mg-regulated genes was challenged by studying their expression after other mineral deficiencies and environmental stresses. The possibility to develop markers for Mg incipient deficiency is discussed here.

Acidic pH shock induces the expressions of a wide range of stress-response genes

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Hong Soon-Kwang

2008-12-01

Full Text Available Abstract Background Environmental signals usually enhance secondary metabolite production in Streptomycetes by initiating complex signal transduction system. It is known that different sigma factors respond to different types of stresses, respectively in Streptomyces strains, which have a number of unique signal transduction mechanisms depending on the types of environmental shock. In this study, we wanted to know how a pH shock would affect the expression of various sigma factors and shock-related proteins in S. coelicolor A3(2. Results According to the results of transcriptional and proteomic analyses, the major number of sigma factor genes were upregulated by an acidic pH shock. Well-studied sigma factor genes of sigH (heat shock, sigR (oxidative stress, sigB (osmotic shock, and hrdD that play a major role in the secondary metabolism, were all strongly upregulated by the pH shock. A number of heat shock proteins including the DnaK family and chaperones such as GroEL2 were also observed to be upregulated by the pH shock, while their repressor of hspR was strongly downregulated. Oxidative stress-related proteins such as thioredoxin, catalase, superoxide dismutase, peroxidase, and osmotic shock-related protein such as vesicle synthases were also upregulated in overall. Conclusion From these observations, an acidic pH shock was considered to be one of the strongest stresses to influence a wide range of sigma factors and shock-related proteins including general stress response proteins. The upregulation of the sigma factors and shock proteins already found to be related to actinorhodin biosynthesis was considered to have contributed to enhanced actinorhodin productivity by mediating the pH shock signal to regulators or biosynthesis genes for actinorhodin production.

NCBI nr-aa BLAST: CBRC-XTRO-01-2512 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-XTRO-01-2512 ref|YP_798849.1| Sphingomyelinase B [Leptospira borgpetersenii se...rovar Hardjo-bovis L550] gb|ABJ79916.1| Sphingomyelinase B [Leptospira borgpetersenii serovar Hardjo-bovis L550] YP_798849.1 8e-06 25% ...

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

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Liu, Yanbin; Koh, Chong Mei John; Ngoh, Si Te; Ji, Lianghui

2015-10-26

Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. The intron 1-containing DAO1 promoters coupled with a DAO1 null

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

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Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

Science.gov (United States)

Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-05-09

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

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Ramesh C. Gupta

2008-03-01

Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

Gene polymorphisms of desaturase enzymes of polyunsaturated fatty acid metabolism and adiponutrin and the increased risk of nonalcoholic fatty liver disease

OpenAIRE

Manvi Vernekar; Deepak Amarapurkar; Kalpana Joshi; Rekha Singhal

2017-01-01

Nonalcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome (MetS). Adiponutrin gene polymorphisms have been associated with NAFLD worldwide. Polyunsaturated fatty acids (PUFAs) have been studied to have anti-inflammatory effects and plasma lipid lowering properties. PUFAs are endogenously synthesized with the help of delta-6-desaturase and delta-5-desaturase enzymes. They are encoded by FADS2 and FADS1 genes respectively. Polymorphisms in ...

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

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Gao Qianqian

2012-03-01

Full Text Available Abstract Background Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria. Results Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS, have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent. Conclusions Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.

Comparative genomics of the lactic acid bacteria

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Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rokhsar, D.; Lucas, S.; Huang, K.; Goodstein, D. M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J. -H.; Diaz-Muniz, I.; Dosti, B.; Smeianov, V; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O' Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D.

2006-06-01

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

Science.gov (United States)

Ohlrogge, John B.; Cahoon, Edgar B.; Shanklin, John; Somerville, Christopher R.

1995-01-01

The present invention relates to a process for producing lipids containing the fatty acid petroselinic acid in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a .omega.12 desaturase from another species which does normally accumulate petroselinic acid.

Effect of Eicosapentaenoic Acid and Docosahexaenoic Acid on Myogenesis and Mitochondrial Biosynthesis during Murine Skeletal Muscle Cell Differentiation

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Tun-Yun Hsueh

2018-03-01

Full Text Available Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA. After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed (P ≤ 0.05 compared with control cells. The expression of the genes related to myogenesis was significantly lower (P ≤ 0.05 in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher (P ≤ 0.05 expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower (P ≤ 0.05 gene expression and lower (P ≤ 0.05 mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher (P ≤ 0.05 in cells treated with fatty acids. Moreover, fatty-acid treatment reduced (P ≤ 0.05 oxygen consumption rate under oligomycin-inhibited (reflecting proton leak and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids