Abstract 373: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 Lowers Tissue Levels of Asymmetric Dimethylarginine and Improves Endothelial Function in Mouse Aortas

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Roman N Rodionov ◽  
Dmitrii V Burdin ◽  
Vladimir Todorov ◽  
Silke Brilloff ◽  
Natalia Jarzebska ◽  
...  
Keyword(s):  
Vascular Function ◽  
Asymmetric Dimethylarginine ◽  
Physiological Role ◽  
Normal Weight ◽  
Maximal Response ◽  
Direct Role ◽  
Developmental Abnormalities ◽  
Vasomotor Function ◽  
Glyoxylate Aminotransferase

Introduction: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, which has been proposed to play a direct role in the pathogenesis of cardiovascular disease. There are two enzymatic pathways for degradation of ADMA: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The first pathway is well characterized, whereas the physiological role of AGXT2 is still poorly understood. The goal of our study was to test the hypothesis that transgenic overexpression of AGXT2 would lead to lowering of systemic levels of ADMA and improved vasomotor function. Methods and results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. Ubiquitous overexpression of the transgene was confirmed by qPCR and Western Blot. TG animals had normal weight and no observed developmental abnormalities. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels in AGXT2 TG animals were decreased by 15% (p<0.05), whereas ADGV levels were 6 times higher in TG animals in comparison with wild-types (p<0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p<0.05). Isolated aortic rings were used to estimate endothelium-dependent and -independent relaxation in response to acetylcholine (Ach) and sodium nitroprusside (SNP), respectively. Aortas from AGXT2 TG mice demonstrated an increase in maximal response to ACh (p<0.05). There was a similar relaxation in response to SNP in both groups. Conclusions: Our findings show that upregulation of AGXT2 results in lower ADMA levels and improved endothelial-dependent relaxation in vivo. AGXT2 thereby may be a therapeutic target for long-term reduction of systemic ADMA levels and improvement of vascular function in vivo. This is especially important, because all the efforts to develop ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far. Our data suggest that AGXT2 might be a promising drug target for cardiovascular pathologies associated with elevated ADMA levels.

Abstract 453: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Natalia Jarzebska ◽  
Roman N Rodionov ◽  
Dmitri Burdin ◽  
Silke Brilloff ◽  
Vladimir T Todorov ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Asymmetric Dimethylarginine ◽  
Vasomotor Function ◽  
Arterial Blood ◽  
Plasma Adma ◽  
Increased Risk ◽  
Glyoxylate Aminotransferase ◽  
Oxide Synthase

Background: Elevation of the endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been shown to be associated with increased risk of cardiovascular diseases. There are two major pathways of ADMA catabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is poorly characterized. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of systemic levels of ADMA and improvement of vasomotor function. Methods and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. There were no developmental or phenotypic changes in the TG animals. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels were decreased by 15% (p<0.05) in the TG mice, whereas ADGV plasma levels were 6 times higher in comparison with wild-types littermates (p<0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p<0.05). TG mice demonstrated improved endothelium-dependent vasodilation (in response to acetylcholine) in aortic rings. The endothelium-independent relaxation (in response to sodium nitroprusside) was unchanged. There was no difference in mean arterial blood pressure measured by telemetry between the wild type and AGXT2 TG mice. In further experiments, we crossed the AGXT2 TG mice with DDAH1 KO mice and showed that upregulation of AGXT2 protects DDAH1 KO mice from elevation of plasma ADMA levels. Conclusion: In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation in vivo. AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.

scholarly journals Endothelial dysfunction and body mass index: is there a role for plasma peroxynitrite?

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Theresa Chikopela ◽  
Douglas C. Heimburger ◽  
Longa Kaluba ◽  
Pharaoh Hamambulu ◽  
Newton Simfukwe ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Body Mass ◽  
Vascular Function ◽  
Aortic Ring ◽  
Normal Weight ◽  
Oxide Synthase ◽  
Weight Groups

Abstract Background Endothelial function is dependent on the balance between vasoconstrictive and vasodilatory substances. The endothelium ability to produce nitric oxide is one of the most crucial mechanisms in regulating vascular tone. An increase in inducible nitric oxide synthase contributes to endothelial dysfunction in overweight persons, while oxidative stress contributes to the conversion of nitric oxide to peroxynitrite (measured as nitrotyrosine in vivo) in underweight persons. The objective of this study was to elucidate the interaction of body composition and oxidative stress on vascular function and peroxynitrite. This was done through an experimental design with three weight groups (underweight, normal weight and overweight), with four treatment arms in each. Plasma nitrotyrosine levels were measured 15–20 h post lipopolysaccharide (LPS) treatment, as were aortic ring tension changes. Acetylcholine (ACh) and sodium nitroprusside (SNP) challenges were used to observe endothelial-dependent and endothelial-independent vascular relaxation after pre-constriction of aortic rings with phenylephrine. Results Nitrotyrosine levels in saline-treated rats were similar among the weight groups. There was a significant increase in nitrotyrosine levels between saline-treated rats and those treated with the highest lipopolysaccharide doses in each of the weight groups. In response to ACh challenge, Rmax (percentage reduction in aortic tension) was lowest in overweight rats (112%). In response to SNP, there was an insignificantly lower Rmax in the underweight rats (106%) compared to the normal weight rats (112%). Overweight rats had a significant decrease in Rmax (83%) in response to SNP, signifying involvement of a more chronic process in tension reduction changes. A lower Rmax accompanied an increase in peroxynitrite after acetylcholine challenge in all weight groups. Conclusions Endothelial dysfunction, observed as an impairment in the ability to reduce tension, is associated with increased plasma peroxynitrite levels across the spectrum of body mass. In higher-BMI rats, an additional role is played by vascular smooth muscle in the causation of endothelial dysfunction.

scholarly journals Pax3-FKHR Knock-In Mice Show Developmental Aberrations but Do Not Develop Tumors

2002 ◽  
Vol 22 (20) ◽  
pp. 7204-7216 ◽  
Author(s):  
Irina Lagutina ◽  
Simon J. Conway ◽  
Jack Sublett ◽  
Gerard C. Grosveld
Keyword(s):  
Germ Line ◽  
Perinatal Death ◽  
Embryonic Stem ◽  
Fibroblast Cell ◽  
Direct Role ◽  
Developmental Defects ◽  
Developmental Abnormalities ◽  
Cdna Sequences ◽  
Valve Insufficiency

ABSTRACT Alveolar rhabdomyosarcoma is a pediatric disease specified by the recurrent chromosome translocations t(2;13) and t(1;13). These translocations result in the formation of the PAX3-FKHR and PAX7-FKHR fusion genes, which are thought to play a causal role in the genesis of this disease. Although PAX3-FKHR exhibits transforming activity in immortalized fibroblast cell lines, a direct role of this fusion protein in tumorigenesis in vivo has not been shown. We determined whether expression of Pax3-FKHR in the mouse germ line would render these animals prone to the development of rhabdomyosarcomas. By targeting FKHR cDNA sequences into the Pax3 locus of embryonic stem cells, we used these cells to generate mice carrying a Pax3-FKHR knock-in allele. Despite low expression of the knock-in allele, heterozygous offspring of Pax3-FKHR chimeric mice showed developmental abnormalities. These included intraventricular septum defects, tricuspid valve insufficiency, and diaphragm defects, which caused congestive heart failure leading to perinatal death. In addition, Pax3-FKHR heterozygous offspring displayed malformations of some but not all hypaxial muscles. However, neither newborn heterozygous pups nor their chimeric parents showed any signs of malignancy. We conclude that the Pax3-FKHR allele causes lethal developmental defects in knock-in mice but might be insufficient to cause muscle tumors.

scholarly journals Characterizing the in vivo role of trehalose inSaccharomyces cerevisiaeusing theAGT1transporter

2015 ◽  
Vol 112 (19) ◽  
pp. 6116-6121 ◽  
Author(s):  
Patrick A. Gibney ◽  
Ariel Schieler ◽  
Jonathan C. Chen ◽  
Joshua D. Rabinowitz ◽  
David Botstein
Keyword(s):  
Large Body ◽  
Physiological Role ◽  
Companion Paper ◽  
Direct Role ◽  
Growth Defects ◽  
Growth Inhibitory ◽  
Trehalose Biosynthesis

Trehalose is a highly stable, nonreducing disaccharide of glucose. A large body of research exists implicating trehalose in a variety of cellular phenomena, notably response to stresses of various kinds. However, in very few cases has the role of trehalose been examined directly in vivo. Here, we describe the development and characterization of a system inSaccharomyces cerevisiaethat allows us to manipulate intracellular trehalose concentrations independently of the biosynthetic enzymes and independently of any applied stress. We found that many physiological roles heretofore ascribed to intracellular trehalose, including heat resistance, are not due to the presence of trehalose per se. We also found that many of the metabolic and growth defects associated with mutations in the trehalose biosynthesis pathway are not abolished by providing abundant intracellular trehalose. Instead, we made the observation that intracellular accumulation of trehalose or maltose (another disaccharide of glucose) is growth-inhibitory in a carbon source-specific manner. We conclude that the physiological role of the trehalose pathway is fundamentally metabolic: i.e., more complex than simply the consequence of increased concentrations of the sugar and its attendant physical properties (with the exception of the companion paper where Tapia et al. [Tapia H, et al. (2015)Proc Natl Acad Sci USA, 10.1073/pnas.1506415112] demonstrate a direct role for trehalose in protecting cells against desiccation).

scholarly journals The Novel Hepatokine Ectodysplasin A Is Increased in Obesity and Reduced after Liraglutide Management

2021 ◽  
Author(s):  
Zhensheng Cai ◽  
Xia Deng ◽  
Panpan Zhang ◽  
Zhicong Zhao ◽  
Chang Guo ◽  
...  
Keyword(s):  
Weight Loss ◽  
Metabolic Diseases ◽  
Liver Lipid ◽  
Physiological Role ◽  
Normal Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Liver Lipid Metabolism ◽  
Mrna And Protein Expression

Abstract Background Ectodysplasin A (EDA), a new hepatokine, has been recently characterized to play a role in liver lipid metabolism and insulin resistance, but its physiological role remains scarcely acquainted in obesity. This study was the first time to determine the level of serum EDA in obesity, and to assess change in the levels of EDA after weight loss in obese mice.Methods We analyzed the serum concentrations of EDA by enzyme-linked immunosorbent assay (ELISA) in 60 subjects including 30 normal weight and 30 obesity. Male C57BL/6J mice were fed with high-fat diet and injected by liraglutide to reduce weight. AML12 cells were induced by palmitic acid and treated with liraglutide. Quantitative real-time PCR and Western blot analysis were conducted to evaluate the expression of EDA. Results Serum EDA levels were significantly higher in obesity than in normal weight subjects. It was positively correlated with body mass index (BMI). More importantly, the mRNA and protein expression of EDA reduced after liraglutide management in vivo and in vitro.Conclusions The level of EDA increased significantly in obesity and decreased significantly after weight loss. It is suggested that EDA may be a novel hepatokine associated with obesity-related metabolic diseases.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

2006 ◽  
Vol 26 (14) ◽  
pp. 5249-5258 ◽  
Author(s):  
Vincenzo Coppola ◽  
Colleen A. Barrick ◽  
Sara Bobisse ◽  
Maria Cecilia Rodriguez-Galan ◽  
Michela Pivetta ◽  
...  
Keyword(s):  
Immune System ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Physiological Role ◽  
Scaffold Protein ◽  
Nucleotide Exchange ◽  
Lymphocyte Migration ◽  
And Migration

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

Noradrenaline-induced calorigenesis in warm- or cold-acclimated rats. In vivo estimation of adrenoceptor concentration of noradrenaline effecting half-maximal response

1980 ◽  
Vol 58 (9) ◽  
pp. 1072-1077 ◽  
Author(s):  
Florent Depocas ◽  
Gloria Zaror-Behrens ◽  
Suzanne Lacelle
Keyword(s):  
Adipose Tissue ◽  
Steady State ◽  
Brown Adipose Tissue ◽  
Maximal Response ◽  
Brown Adipose ◽  
Acclimation Group ◽  
Arterial Plasma ◽  
State Concentration ◽  
Na Uptake

Desmethylimipramine (DMI, 1 mg DMI∙HCl kg−1) and normetanephrine (NMN, 1 μg min−1 g−0.74) were used to inhibit, respectively, neuronal and extraneuronal uptakes of noradrenaline (NA) during calorigenesis induced in barbital-sedated warm-acclimated (WA) or cold-acclimated (CA) rats by infusion of NA, a procedure which mimics the effects of NA released within calorigenic tissues in response to cold exposure. The doses of the inhibitors were selected for maximal effectiveness in potentiating calorigenic response and for minimal side effects. For rats of either acclimation group treated with DMI and NMN, with DMI only, or with neither inhibitor the doses of NA required to evoke approximately half-maximal calorigenic responses were, respectively, 0.5, 1.0, and 3.5 ng min−1 g−0.74. The corresponding steady-state concentrations of NA in arterial plasma averaged 14.3, 21.7, and 43.2 nM in the three groups of WA rats and 10.0, 14.8, and 31.9 nM in the three groups of CA rats. Reduction by NA uptake inhibitors of the circulating levels of NA necessary to stimulate calorigenesis, half-maximally, presumably in brown adipose tissue, indicates a reduction in the steepness of the NA concentration gradient between capillary plasma and synaptic clefts in that tissue. The steady-state concentration of NA in blood plasma of rats treated with DMI and NMN and infused with NA at a dose of 0.5 ng min−1 g−0.74 (~1 × 10−8 M) is a good estimate of the NA concentration required at calorigenic adrenoceptors to effect half-maximal activation. Presumably, this concentration is also an estimate of that resulting from NA released at nerve endings during cold-induced activation of nonshivering thermogenesis at half-maximal rates in brown adipose tissue.

scholarly journals Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria

Open Biology ◽  
2017 ◽  
Vol 7 (7) ◽  
pp. 170087 ◽  
Author(s):  
Yi Ting Tsai ◽  
Valentina Salzman ◽  
Matías Cabruja ◽  
Gabriela Gago ◽  
Hugo Gramajo
Keyword(s):  
Cell Envelope ◽  
Phospholipid Composition ◽  
Mycolic Acid ◽  
Transcriptional Level ◽  
Direct Role ◽  
Conditional Mutant ◽  
In The Wild ◽  
Fas Ii

One of the dominant features of the biology of Mycobacterium tuberculosis , and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc 2 155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and P fasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C 18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

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