Arginine Catabolism Research Articles

RrmAregulates the stability of specific transcripts in response to both nitrogen source and oxidative stress

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′ UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

Inhibition of endogenous urease activity by NBPT application reveals differential N metabolism responses to ammonium or nitrate nutrition in pea plants: a physiological study

Urea is the predominant form of N applied as fertilizer to crops, but it is also a significant N metabolite of plants themselves. As such, an understanding of urea metabolism in plants may contribute significantly to subsequent N fertilizer management. It currently appears that arginase is the only plant enzyme that can generate urea in vivo. The aim of this work was, therefore, to gain a more in-depth understanding of the significance of the inhibition of endogenous urease activity and its role in N metabolism depending on the N source supplied. Pea (Pisum sativum cv. Snap-pea) plants were grown with either ammonium or nitrate as the sole N source in the presence or absence of the urease inhibitor NBPT. When supplied, NBPT is absorbed by plants and translocated from the roots to the leaves, where it reduces endogenous urease activity. Different N metabolic responses in terms of N-assimilatory enzymes and N-containing compounds indicate a different degree of arginine catabolism activation in ammonium- and nitrate-fed plants. The arginine catabolism is more highly activated in ammonium-fed plants than in nitrate-fed plants, probably due to the higher turnover of substrates by enzymes playing a key role in N recycling and remobilization during catabolism and in early flowering and senescence processes, usually observed under ammonium nutrition.

Increased Erythrocytes By-Products of Arginine Catabolism Are Associated with Hyperglycemia and Could Be Involved in the Pathogenesis of Type 2 Diabetes Mellitus

Diabetes mellitus (DM) is a worldwide disease characterized by metabolic disturbances, frequently associated with high risk of atherosclerosis and renal and nervous system damage. Here, we assessed whether metabolites reflecting oxidative redox state, arginine and nitric oxide metabolism, are differentially distributed between serum and red blood cells (RBC), and whether significant metabolism of arginine exists in RBC. In 90 patients with type 2 DM without regular treatment for diabetes and 90 healthy controls, paired by age and gender, we measured serum and RBC levels of malondialdehyde (MDA), nitrites, ornithine, citrulline, and urea. In isolated RBC, metabolism of L-[14C]-arginine was also determined. In both groups, nitrites were equally distributed in serum and RBC; citrulline predominated in serum, whereas urea, arginine, and ornithine were found mainly in RBC. DM patients showed hyperglycemia and increased blood HbA1C, and increased levels of these metabolites, except for arginine, significantly correlating with blood glucose levels. RBC were observed to be capable of catabolizing arginine to ornithine, citrulline and urea, which was increased in RBC from DM patients, and correlated with an increased affinity for arginine in the activities of putative RBC arginase (Km = 0.23±0.06 vs. 0.50±0.13 mM, in controls) and nitric oxide synthase (Km = 0.28±0.06 vs. 0.43±0.09 mM, in controls). In conclusion, our results suggest that DM alters metabolite distribution between serum and RBC, demonstrating that RBC regulate serum levels of metabolites which affect nitrogen metabolism, not only by transporting them but also by metabolizing amino acids such as arginine. Moreover, we confirmed that urea can be produced also by human RBC besides hepatocytes, being much more evident in RBC from patients with type 2 DM. These events are probably involved in the specific physiopathology of this disease, i.e., endothelial damage and dysfunction.

Hot air treatment‐induced arginine catabolism is associated with elevated polyamines and proline levels and alleviates chilling injury in postharvest tomato fruit

To understand whether arginine catabolism might be involved in hot air (HA)-induced chilling tolerance mechanism in tomato fruit, we investigated the effect of HA treatment on endogenous arginine catabolism in relation to chilling injury. Tomato fruit were harvested at mature green stage and treated with HA at 38°C for 12 h and then stored at 2°C for 21 days. The effects of HA treatment on fruit chilling injury and gene expression levels or enzyme activity, and metabolites related to arginine catabolism were evaluated. HA treatment reduced the chilling injury symptoms of tomato fruit and enhanced the accumulation of endogenous polyamines, especially putrescine and proline. This accumulation is associated with the increased transcript levels of genes encoding arginase (LeARG1 and LeARG2), arginine decarboxylase (LeADC), ornithine decarboxylase (LeODC) and ornithine aminotransferase (LeOAT) at most sampling times. However, HA treatment had little effect on nitric oxide synthase activity and nitric oxide concentration. These results revealed that the reduction in chilling injury by HA treatment may be due to the accumulation of putrescine and proline induced primarily by activating the catabolism of endogenous arginine.

Depletion of activated lymphocytes during the emergence of ovarian cancer (P2004)

Abstract We are modeling the myeloid suppression found in tumors by characterizing how peritoneal cavity (PerC) macrophages (Mϕs) suppress lymphocyte activation. Cultured PerC Mϕs suppress the proliferation of C57BL/6J T cells by iNOS catabolism of arginine. To make our model more relevant to in vivo biology we initiated studies of ovarian cancer in the PerC. The ID8 cell line (mouse ovarian surface epithelial cell carcinoma) forms ovarian tumors and hemorrhagic ascites 5 weeks following i.p. injection into intact, syngeneic C57BL/6J mice. We found cancer development was accelerated in IL10-/- mice and delayed in iNOS-/- mice indicative that inflammation plays a role in cancer progression. Assessing mice before the ovarian cancer was fully developed we observed a depletion of CD44hi T and CD11b+IgMhi B cells in the PerC. With disease progression B and T cell depletion also became evident in the spleen. Lymphocyte depletion in the PerC correlated with ID8 expansion and the emergence of CD11b+F4/80+Gr-1+CD80+ myeloid suppressor cells (MSCs); splenic MSCs were CD80-. It was interesting to observe that GITR+ T cells, naturally enriched in the PerC of healthy, normal mice, persisted during MSC emergence in both the spleen and PerC. We are investigating what triggers the selective death of these important lymphocyte subsets. These results reveal an additional, challenging aspect of the immune suppression characteristic of myeloid cell emergence during cancer development.

In vivo role of Arabidopsis arginase in arginine metabolism and abiotic stress response

Nitric oxide (NO) and polyamines play essential roles in many developmental processes and abiotic stress responses in plants. NO and polyamines are metabolized from arginine through NO synthase (NOS) and arginine decarboxylase (ADC), respectively. Function of arginase, another important enzyme involved in arginine metabolism, in abiotic stress remains largely unknown. In the recent study, we have dissected the impact of arginase on arginine metabolism and abiotic stress responses through manipulating AtARGAHs expression. The results suggested that manipulation of arginase expression modulated accumulation of arginine and direct downstream products of arginine catabolism. AtARGAHs knockout lines exhibited increased accumulation of polyamines and NO and enhanced abiotic stress tolerance, while AtARGAHs overexpressing lines displayed the opposite results. Notably, we highlighted that Arabidopsis arginase plays distinctive and dual roles in the crosstalk between polyamines and NO signaling during abiotic stress responses, mediating both arginine metabolism and reactive oxygen species (ROS) accumulation. It is likely that accumulation of both NO and polyamines might activate abiotic stress responses in the plant.

Abstract 3714: Metabolomics and proteomics reveal inhibition of glycolysis in withaferin A-mediated prevention of mammary carcinogenesis in MMTV-neu mice.

Abstract Plants used in Ayurvedic medicine, which has been practiced for centuries in India for the treatment of different ailments, continue to draw attention for identification of novel small molecule cancer chemopreventive agents. Withaferin A (WA) is one such promising small molecule constituent of the medicinal plant Withania somnifera (also known as Ashwagandha) with in vivo activity against xenografted human breast cancer cells. We have also shown recently that mammary cancer development and pulmonary metastasis in MMTV-neu transgenic mice is inhibited significantly by WA administration. In the present study, we performed metabolomics profiling using plasma and tumor tissues (n=8) and proteomics profiling using tumor tissues (n=3) from control and WA-treated MMTV-neu mice to gain insights into the mechanism underlying in vivo preventive effect of WA. A total of 320 and 328 biochemicals of known identity were detectable in the plasma and tumor tissue, respectively. The WA-mediated mammary cancer prevention in MMTV-neu mice was associated with a statistically significant alteration (P≤0.05) in the levels of 76 biochemicals in the plasma (24 increase, 52 decrease). Metabolomics using tumor tissues revealed alterations in the levels of 24 biochemicals (2 increase, 24 decrease) upon WA administration. One change that was highly consistent in the plasma and tumor tissue from WA-treated mice compared to controls was a decrease in glucose utilization, glycolysis, and TCA cycle activity. Proteomics using tumor tissues confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase A isoform 2. Downregulation of TCA cycle-related proteins in the tumor of WA-treated mice compared with control included pyruvate carboxylase and isocitrate dehydrogenase. Other altered biochemicals were reflective of changes in arginine metabolism, polyamine biosynthesis, glutathione metabolism, lipid metabolism, and catabolism of branched-chain amino acids. These observations indicate that WA treatment prevents mammary cancer in MMTV-neu mice by suppressing glycolysis (Warburg effect) and TCA cycle. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Shivendra V. Singh, Eun-Ryeong Hahm, Joomin Lee, Su-Hyeong Kim, Anuradha Sehrawat, Julie A. Arlotti. Metabolomics and proteomics reveal inhibition of glycolysis in withaferin A-mediated prevention of mammary carcinogenesis in MMTV-neu mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3714. doi:10.1158/1538-7445.AM2013-3714

An Evolutionary Firefly Algorithm for the Estimation of Nonlinear Biological Model Parameters

The development of accurate computational models of biological processes is fundamental to computational systems biology. These models are usually represented by mathematical expressions that rely heavily on the system parameters. The measurement of these parameters is often difficult. Therefore, they are commonly estimated by fitting the predicted model to the experimental data using optimization methods. The complexity and nonlinearity of the biological processes pose a significant challenge, however, to the development of accurate and fast optimization methods. We introduce a new hybrid optimization method incorporating the Firefly Algorithm and the evolutionary operation of the Differential Evolution method. The proposed method improves solutions by neighbourhood search using evolutionary procedures. Testing our method on models for the arginine catabolism and the negative feedback loop of the p53 signalling pathway, we found that it estimated the parameters with high accuracy and within a reasonable computation time compared to well-known approaches, including Particle Swarm Optimization, Nelder-Mead, and Firefly Algorithm. We have also verified the reliability of the parameters estimated by the method using an a posteriori practical identifiability test.

Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical () concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding.

Structural determinants of substrate specificity in aldehyde dehydrogenases

Within the aldehyde dehydrogenase (ALDH) superfamily, proteins belonging to the ALDH9, ALDH10, ALDH25, ALDH26 and ALDH27 families display activity as ω-aminoaldehyde dehydrogenases (AMADHs). These enzymes participate in polyamine, choline and arginine catabolism, as well as in synthesis of several osmoprotectants and carnitine. Active site aromatic and acidic residues are involved in binding the ω-aminoaldehydes in plant ALDH10 enzymes. In order to ascertain the degree of conservation of these residues among AMADHs and to evaluate their possible relevance in determining the aminoaldehyde specificity, we compared the known amino acid sequences of every ALDH family that have at least one member with known crystal structure, as well as the electrostatic potential surface of the aldehyde binding sites of these structures. Our analyses showed that four or three aromatic residues form a similar “aromatic box” in the active site of the AMADH enzymes, being the equivalents to Phe170 and Trp177 (human ALDH2 numbering) strictly conserved in all of them, which supports their relevance in binding the aminoaldehyde by cation–π interactions. In addition, all AMADHs exhibit a negative electrostatic potential surface in the aldehyde-entrance tunnel, due to side-chain carboxyl and hydroxyl groups or main-chain carbonyl groups. In contrast, ALDHs that have non-polar or negatively charged substrates exhibit neutral or positive electrostatic potential surfaces, respectively. Finally, our comparative sequence analyses revealed that the residues equivalent to Asp121 and Phe170 are highly conserved in many ALDH families irrespective of their substrate specificity—suggesting that they perform a role in catalysis additional or different to binding of the substrate—and that the positions Met124, Cys301, and Cys303 are hot spots changed during evolution to confer aldehyde specificity to several ALDH families.

Amelioration of chilling stress by arginine in tomato fruit: Changes in endogenous arginine catabolism

Freshly harvested tomato fruit were pretreated with 0.2mM arginine at −35kPa for 0.5min and then stored at 2°C for 28d to investigate the effect of exogenous arginine treatment on endogenous arginine catabolism in relation to chilling injury (CI). Arginine treatment reduced the CI index of fruit and enhanced accumulation of polyamines, especially putrescine, and proline, which resulted from the increased activities of the catabolic enzymes arginase, arginine decarboxylase, ornithine decarboxylase and ornithine δ-aminotransferase at most sampling times. Nitric oxide synthase activity was also increased by arginine treatment, which at least partly contributed to the increased nitric oxide concentration. These results revealed that the reduction in CI by exogenous arginine may be due to the accumulation of putrescine, proline and nitric oxide induced by activating the different pathways of endogenous arginine catabolism.

Factors Associated with Alkali Production from Arginine in Dental Biofilms

Alkali production by oral bacteria in the oral cavity has been linked to protection against dental caries. The current study assessed various parameters associated with ammonium produced during arginine catabolism in dental biofilms. Polymicrobial biofilms were formed with saliva as the inoculum. The NH3 level and the pH of the spent medium were used to monitor and quantitate the bacterial reactions. The presence of sucrose, a low buffer capacity, and a low pH (≤ pH 4.5) were found to hamper alkali production from arginine. The rate of alkali production exhibited an optimum around pH 5.5. Biofilms were found to produce NH3 also from polypeptides and proteins in the medium. The biofilm age affected these processes. The experimental model proved valuable for the assessment of the collective bacterial reactions determining the overall pH outcome. This experimental approach could bridge the gap in our knowledge between pH-rise phenomena and caries susceptibility from clinical observations and studies performed on alkali-producing bacteria in well- controlled, though simplified, in vitro models. Analysis of our data supports the hypothesis that the initiation and progression of dental caries may be influenced by the relative rates of acid and base formation, which critically depend on the aforementioned parameters.

Effect of ethanol and low pH on citrulline and ornithine excretion and arc gene expression by strains of Lactobacillus brevis and Pediococcus pentosaceus

The accumulation of citrulline and ornithine in wine or beer as a result of the arginine catabolism of some lactic acid bacteria (LAB) species increases the risk of ethyl carbamate and putrescine formation, respectively. Several LAB species, which are found as spoilage bacteria in alcoholic beverages, have been reported to be arginine degrading. This study evaluates the effect of ethanol content and low pH on the excretion of citrulline and ornithine by two strains belonging to the potential contaminant species Lactobacillus brevis and Pediococcus pentosaceus. In the conditions that most affected cell viability, arginine consumption per cell increased noticeably, indicating that arginine utilization may be a stress responsive mechanism. L. brevis showed a higher accumulation of ornithine in the media than P. pentosaceus. In the presence of ethanol, a higher expression of the arcC gene was found in P. pentosaceus, which resulted in a lower excretion of citrulline and ornithine than in L. brevis. This suggests that L. brevis is more likely to produce these amino acids, which are precursors of ethyl carbamate and putrescine.

Investigation into the Physiologies of Aeromonas veronii in vitro and Inside the Digestive Tract of the Medicinal Leech Using RNA-seq

Host-associated microbial communities are widespread in nature and vital to the health and fitness of the host. Deciphering the physiology of the microbiome in vivo is critical to understanding the molecular basis of the symbiosis. Recently, the development and application of high-throughput sequencing techniques, particularly RNA-seq, for studying microbial communities has enabled researchers to address not only which microbes are present in a given community but also how the community functions. For microbes that can also be cultivated in the laboratory, RNA-seq provides the opportunity to identify genes that are differentially expressed during symbiosis by comparing in vitro to in vivo transcriptomes. In the current study, we used RNA-seq to identify genes expressed by the digestive-tract microbiome of the medicinal leech, Hirudo verbana, and by one of the two dominant symbionts, Aeromonas veronii, in a rich medium. We used a comparative approach to identify genes differentially expressed during symbiosis and gain insight into the symbiont's physiology in vivo. Notable findings include evidence for the symbionts experiencing environmental stress, performing arginine catabolism, and expressing noncoding RNAs that are implicated in stationary phase survival, a state in which A. veronii persists for months within the host.

PDL1 is not needed for peritoneal macrophage suppression of T cell activation (176.1)

Abstract The myeloid suppression found in tumors can be modeled by studying peritoneal cavity (PerC) macrophages (Mϕs). The high % of Mϕs in the PerC inhibit the proliferation of C57BL/6J (WT) T cells by iNOS catabolism of arginine. PerC T cells from IFNγR-/- mice proliferate indicating that IFNγ drives suppression. IFNγ increases expression of molecules that can promote (B7) or inhibit (B7H1/PDL1) T cell activation. PDL1 was of particular interest as it is upregulated on Mϕs & has been reported to restrict T cell activation in tumors. B7-/-, PDL1-/-, & IFNγR-/- PerC cells were compared to those from WT mice. B7-/- mice had fewer CD4 & CD8 PerC T cells than WT. IFNγR-/- mice had fewer CD4 cells & more CD8 T cells than WT. PDL1-/- mice had fewer CD44hi & CD25+ CD4 T cells & more CD8 T cells, particularly CD44hi & GITR+ cells. B7-/- PerC T cells, like those of IFNγR-/- mice, were less suppressed & responded with PGE2 or IL-10 neutralization. However, they differed in that IL4, via arginase, regulated T cell proliferation in IFNγR-/- mice. Surprisingly like WT mice, PDL1-/- PerC T cells were suppressed by IFNγ-triggered iNOS. Although PDL1 costimulates IL10 production the absence of PDL1 did not increase IFNγ production. These results reveal a challenge for strategies intent on recovering immunity by blocking PDL1 in Mϕ-rich tumor microenvironments.

The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source

The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.

Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea

Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2, catalysing the catabolism of arginine into ornithine and urea. Arginine and ornithine are both precursors for polyamine biosynthetic pathways. We observed an accumulation of ARGAH2 mRNA in Arabidopsis upon inoculation with the necrotrophic pathogen Botrytis cinerea. Transgenic lines displaying either overexpression of ARGAH2 or simultaneous silencing of both Arabidopsis arginase-encoding genes were created and their resistance to B. cinerea infection evaluated. Overexpression of arginase resulted in changes in amino acid accumulation, while polyamine levels remained largely unaffected. Silencing lines were affected in both amino acid and putrescine accumulation. Arabidopsis plants overexpressing the arginase gene were less susceptible to B. cinerea, whereas silencing lines remained as susceptible as the wild type. We discuss how arginase might interact with plant defence mechanisms. These results provide new insights into amino acid metabolic changes under stress.

Clinical and Genetic Variability of Red Blood Cell Hemolysis in Sickle Cell Anemia

Abstract 1077Intravascular hemolysis is an important pathological mechanism underlying some complications of sickle cell disease and other hemolytic anemias. Hemolysis contributes to endothelial dysfunction, pulmonary and systemic vasculopathy, and platelet and hemostatic activation via nitric oxide catabolism by plasma hemoglobin and arginine catabolism by red blood cell arginase. Little is known about the molecular mechanisms of hemolysis and how the propensity of erythrocytes to hemolyze is modulated. Hemoglobin F concentration and the presence of ∝ thalassemia affect the level of hemolysis but it is likely that other genes and their products are also important. We hypothesize that genetic variation, much of which is outside the β-globin gene-like cluster, underlies the susceptibility of erythrocytes to hemolyze in response to diverse disease stressors.We first characterized hemolysis by creating a principal component analysis (PCA) of age-adjusted values for LDH, AST, reticulocyte count and total bilirubin, but not hemoglobin concentration, to develop a hemolytic component that reflects shared variability among markers. The development of such a component helps to resolve the problem of dealing with correlated predictors in multivariate analyses and confounding variables such as site, and it permits for adjustment for the degree of anemia. To validate the PCA, we measured the plasma hemoglobin levels and red cell microparticle levels in the first and fourth quartile of PCA intensity of hemolysis in 118 HbS-only patients without detectable HbA, from the Walk-PHASST cohort We observed a highly significant increase in plasma hemoglobin (p<0.0001) and red cell microparticles (p=0.0004) based on PCA quartile. Despite the small sample size of this validating cohort we reproduced significant associations between high hemolytic rate and the subphentypes of low arterial oxygen saturation, high pulse pressure, leg ulcers, TRV, high NT-proBNP levels, and low 6-minute walk test distance. More patients with ∝ thalassemia and more females were present in the lower hemolytic index quartile (p=0.006).The hemolytic index and its individual components were then used as phenotypes in genome-wide association studies (GWAS) in the CSSCD (Cooperative Study of Sickle Cell Disease) and walk-PHaSST cohorts to discover novel genes that might be associated with hemolysis. As further validation of our approach using PCA stratification, patients in the quartile with the lowest hemolytic index from the CSSCD also had a much higher prevalence of ∝ thalassemia than patients within the highest quartile of hemolytic index (p=2.2E-16). We first examined 1117 cases from the CSSCD and found 303 SNPs, 265 with a MAF >0.05, that reached a threshold of significance of p<5E-4. For replication, we examined these SNPs in the Walk-PHASST cohort. Eight SNPs replicated with the same effects in a GWAS in 449 subjects from Walk-PHAAST and p-value<0.01. Of the 8 SNPs that replicated, 4 SNPs were in olfactory receptor (OR) genes on chromosome (chr) 11p; OR51L2 (rs7948471, rs7938426. rs1391617), and OR51L1 (rs2445284). Several of these SNPs were also associated with HbF in previous GWAS analyses. Polymorphisms in the OR gene cluster upstream of HBG might modulate HbF levels by altering chromatin structure within the HBB globin gene-like cluster. One SNP in an intron of NPRL3 (rs7203566) on chr16p is ∼34 kb upstream from a SNP causing ∝ thalassemia. In CSSCD cases there was an association of SNPs in NPRL3 with reticulocytes (p=5.1E-0006) and LDH (p=0.0003). In silico analysis did not predict any function for this SNP.Genetic studies to discover new biologic modifiers of hemolysis will help to identify critical molecular determinants of hemolysis for functional studies, to develop new disease severity biomarkers, and to suggest candidate therapies for some common human diseases with intravascular hemolysis. We anticipate that our studies will identify genetic variants enriched in the African-American population primarily determined by the evolved human response to endemic malaria infection. These studies are expected to broadly impact many human diseases and blood banking by providing genomic markers of susceptibility to hemolytic anemia, red cell storage stability and transfusion risk, and insights into novel strategies to reduce anemia and to enhance red blood cell storage and post-transfusion erythrocyte recovery. Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties.

Nitric oxide synthase is not expressed, nor up-regulated in response to cold acclimation in liver or muscle of threespine stickleback (Gasterosteus aculeatus)

There are three isoforms of the enzyme nitric oxide synthase (NOS) in mammals: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). All three isoforms oxidize arginine to citrulline in a reaction producing nitric oxide (NO), which regulates multiple signaling pathways and physiological functions in mammals. Less is known about NOS in fishes, in which the existence of eNOS is controversial. Nevertheless, multiple adjustments occur during cold acclimation of fishes, several of which are known to be mediated by eNOS and NO in mammals, including mitochondrial biogenesis, vasodilation and angiogenesis. We hypothesized that if NOS was present, and NO stimulated these pathways in fishes, then the activity of NOS would increase during cold acclimation. To test this hypothesis, we measured the activity and mRNA levels of NOS in three tissues (liver, oxidative muscle, glycolytic muscle) known to undergo mitochondrial biogenesis and/or angiogenesis. Measurements were made in the threespine stickleback, Gasterosteus aculeatus acclimated to either warm (20°C) or cold (8°C) temperature for 9weeks. Cold-acclimated fish were harvested on days 1–3, and at weeks 1, 4 and 9 at 8°C, while warm-acclimated fish were harvested on day 0 and after 9 weeks at 20°C. Transcript levels of NOS were quantified using quantitative real-time PCR, and NOS activity was measured using a radiochemical assay, which detected the rate of catabolism of 14C-labeled arginine. Neither NOS activity nor transcripts were detected in oxidative muscle or glycolytic muscle of warm- or cold-acclimated stickleback, although transcript levels of nNOS and NOS activity were detected in brain. Arginine catabolism was detected in liver of animals held at 10°C and 20°C for 9weeks, but was due to arginase activity, rather than NOS. Consistent with this, NOS transcripts were undetectable in liver. The absence of NOS in liver and muscles of stickleback indicates that signaling molecules other than NO likely mediate physiological changes during cold acclimation in stickleback.

Methyl jasmonate alters arginine catabolism and improves postharvest chilling tolerance in cherry tomato fruit

The influence of methyl jasmonate (MeJA) on levels of gene transcripts, enzyme activities and metabolites related to arginine catabolism and chilling injury (CI) was studied in cherry tomato ( Solanum lycopersicum L. cv. Messina) fruit stored at 2 °C for 21 days. The CI index of fruit pretreated with 0.05 mM MeJA vapor for 12 h at 20 °C was reduced compared with that of untreated fruit. The reduction was associated with up-regulated arginine catabolism. The mRNA levels and activities of arginase, arginine decarboxylase (ADC) and ornithine aminotransferase (OAT) in treated fruit were higher than in control fruit, while those of ornithine decarboxylase (ODC) increased transiently in response to MeJA treatment during the early days of cold storage. Free putrescine (Put) and proline accumulated in MeJA-treated fruit, while levels of other arginine-related amino acids were affected by MeJA. Spermidine (Spd) and spermine (Spm) contents remained unchanged in response to MeJA treatment over most of the storage time. The results indicate that MeJA becomes involved in coordinated catabolism of arginine, and helps to improve chilling tolerance in cherry tomato fruit.