Nitric Oxide Synthesis Pathway Research Articles

A lupin protein hydrolysate protects the central nervous system from oxidative stress in WD-fed ApoE-/- mice.

Oxidative stress plays a crucial role in neurodegenerative diseases like Parkinson's and Alzheimer's. Studies indicate the relationship between oxidative stress and the brain damage caused by a high-fat diet. It is previously found that a lupin protein hydrolysate (LPH) has antioxidant effects on human leukocytes, as well as on the plasma and liver of Western diet (WD)-fed ApoE-/- mice. Additionally, LPH shows anxiolytic effects in these mice. Given the connection between oxidative stress and anxiety, this study aimed to investigate the antioxidant effects of LPH on the brain of WD-fed ApoE-/- mice. LPH (100 mg kg-1) or a vehicle is administered daily for 12 weeks. Peptide analysis of LPH identified 101 amino acid sequences (36.33%) with antioxidant motifs. Treatment with LPH palliated the decrease in total antioxidant activity caused by WD ingestion and regulated the nitric oxide synthesis pathway in the brain of the animals. Furthermore, LPH increased cerebral glutathione levels and the activity of catalase and glutathione reductase antioxidant enzymes and reduced the 8-hydroxy-2'-deoxyguanosine levels, a DNA damage marker. These findings, for the first time, highlight the antioxidant activity of LPH in the brain. This hydrolysate could potentially be used in future nutraceutical therapies for neurodegenerative diseases.

MaNCP1, a C2H2 Zinc Finger Protein, Governs the Conidiation Pattern Shift through Regulating the Reductive Pathway for Nitric Oxide Synthesis in the Filamentous Fungus Metarhizium acridum.

ABSTRACTAsexual sporulation is the most common reproduction mode of fungi. Most filamentous fungi have two conidiation patterns, normal conidiation and microcycle conidiation, which may be regulated by nutritional conditions. Nitrogen source can affect the fungal conidiation pattern, but the regulatory mechanism is not fully understood. In this study, we report a C2H2 zinc finger protein, MaNCP1, which has typical transcription factor characteristics and is screened from the subtractive library regulated by nitrate in the entomopathogenic fungus Metarhizium acridum. MaNCP1 and its N-terminal play critical roles in the conidiation pattern shift. Further study shows that MaNCP1 interacts with MaNmrA, which also contributes to the conidiation pattern shift and is involved in the reductive pathway of nitric oxide (NO) synthesis. Intriguingly, the conidiation pattern of the MaNCP1-disruption strain (ΔMaNCP1) can be restored to microcycle conidiation when grown on the microcycle conidiation medium, SYA, supplemented with NO donor or overexpressing MaNmrA in ΔMaNCP1. Here, we reveal that MaNCP1 governs the conidiation pattern shift through regulating the reductive synthesis of NO by physically targeting MaNmrA in M. acridum. This work provides new mechanistic insights into how changes in nitrogen utilization are linked to the regulation of fungal morphological changes.IMPORTANCE Fungal conidia play important roles in the response to environmental stimuli and evasion of the host immune system. The nitrogen source is one of the main factors affecting shifts in fungal conidiation patterns, but the regulatory mechanism involved is not fully understood. In this work, we report that the C2H2 zinc finger protein, MaNCP1, governs the conidiation pattern shift in M. acridum by targeting the MaNmrA gene, thereby altering the regulation of the reductive pathway for NO synthesis. This work provides further insights into how the nutritional environment can regulate the morphogenesis of filamentous fungi.

The role of nitric oxide on the antiarrhythmic effects of ketamine/xylazine in a rat model of acute cardiac ischemia-reperfusion.

The prevalence of ventricular arrhythmias during general anesthesia is about 70%. In experimental studies on the antiarrhythmic effects of different agents, using anesthetic drugs that do not have any protective properties are preferable. The present study was conducted to investigate molecular mechanisms involved in the antiarrhythmic effects of ketamine/xylazine (K/X).Sixty male rats were assigned to eight groups: K/X, L -NAME (25–35 mg/kg) with thiopental (TP), L-NAME (25–35 mg/kg) with ketamine/xylazine, L arginine (100 mg/kg) with thiopental, L-arginine (100 mg/kg) with ketamine/xylazine. After anesthetic induction using TP or K/X, the animals were subjected to 30 min of ischemia. Hemodynamic parameters, ventricular arrhythmias during ischemia, the incidence of ventricular tachycardia (VT), and ventricular fibrillation (VF) were measured. Additionally, in order to assess nitrite/nitrate ratio and LDH after ischemia, serum samples were collected and used.Our results showed that in the K/X group, the number of VT and VF, duration of VT (p = 0.006), the severity of arrhythmias (p = 0.0179). There was no VF incidence in this group.These protective effects were faded by administration of L-NAME with K/X. The combination of L- Arginine in the TP group decreased the number and duration of VT (p < 0.001, p = 0.0013) with no incidence of VF in comparison with TP. L-arginine with K/X groups increased the number and duration of VT (p < 0.0001, p < 0.001) compared to K/X and VF was seen (100%). However, there was no significant difference between TP and K/X groups in terms of this nitrite/nitrate ratio. These findings suggest that the antiarrhythmic effects of ketamine/xylazine might be partially relative to the nitric oxide synthesis pathway.

Nitric Oxide Inhibition Assay and the Respective Target Identification of an Aptamer Designed to Control Atherosclerosis.

Aptamers are emerging newer therapeutics and diagnostics whichcan be designed to bind any kind of target proteins. Vascular endothelial damage by the excess amount of nitric oxide production in systemic circulation leads to the secretion of inflammatory chemoattractants and cell adhesions are the prime pro-atherogenic events in the formation of plagues at atrial intimal layers due to oxidation - sensitive mechanisms. Nitric oxide inhibition assay is one of the valuable qualitative anti-atherosclerosis matrices. In this research, Nitric oxide inhibition efficiency of an ssDNA aptamer on cell lines was studied, and the respective targets of that aptamer were identified by network analysis. The aptamer used here was originally designed for Selectin P Ligand Protein to control the atherogenic process. 20 nM of aptamer solution in LipofectamineTM 2000 sshowshighest level 70.5% inhibition of nitric oxide liberation on 24 hours cultured medium of lipopolysaccharide stimulated murine macrophage RAW 264.7 cell lines. Protein interaction network analysis of the nitric oxide synthesis pathway interactors and the molecular docking analysis with network resulted in proteins such as AKT serine/threonine kinase 1, calmodulin, estrogen receptor 1, and nitric oxide synthase-3, which confirmed that the G - quadruplex Model of 18-mer sequence effectively bound to the active sites of estrogen receptor 1, and nitric oxide synthase-3. The aptamer designed for atherosclerotic target has also exertedsignificant nitric oxide inhibition to control the atherogenic events through the proteins such as, AKT1, NOS3 and ESR1.

Evidence for an arginine-dependent route for the synthesis of NO in the model filamentous fungus Aspergillus nidulans.

Nitric oxide (NO) is a signalling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving l-arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of l-arginine to liquid media elicited a burst of NO that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian NO synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates.

Nω-nitro-L-arginine, a nitric oxide synthase inhibitor, attenuates nickel-induced neurotoxicity

The various mediums of exposure to nickel (Ni) compounds have raised enormous public health concerns, as it has been illustrated to exert toxic effects in biological organs, including the brain. We have previously implicated the involvement of elevated nitric oxide (NO) in Ni-induced oxidative stress in the brain. Hence, the present study investigated the ameliorative potential of Nω-nitro-L-arginine (L-NA), a NO synthase inhibitor, following Ni-induced neurotoxicity. Adult male rats were divided into four groups; control (normal saline), 10 mg/kg Ni chloride (NiCl2) only, 1 mg/kg L-NA, or 2 mg/kg L-NA co-administered with NiCl2. The administration was via daily intraperitoneal injections for three weeks. Neurobehavioural assessments performed thereafter ascertained short-term spatial memory and anxiety. Furthermore, histological evaluations of the cortex, hippocampus, and striatum were carried out using routine hematoxylin and eosin technique, while the phosphotungstic acid hematoxylin method was used to express the degree of astrogliosis. Biochemical analysis of NO levels was examined along with other oxidative stress markers (superoxide dismutase, catalase, glutathione, glutathione S transferase, glutathione peroxidase, myeloperoxidase, and lipid peroxidation). The results illustrated altered behavioral responses, a higher population of degenerating neurons, and astrocytes in the NiCl2 group. There was also an elevation in the NO level and a corresponding reduction in antioxidant activities. However, these debilitating changes were ameliorated in the L-NA treated groups. These results demonstrate an association between alterations in NO synthesis pathway and Ni neurotoxicity, which may render neuronal cells susceptible to damage by oxidative stress. This may yet be another mechanism and useful therapeutic marker in deciphering Ni-induced neurotoxicity.

Presynaptic NK1 Receptor Activation by Substance P Suppresses EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex

Substance P (SP) regulates inhibitory synaptic transmission mediated by GABAA receptors in the cerebral cortex; however, SP-mediated regulation of excitatory synaptic transmission remains poorly understood. We performed whole-cell patch-clamp recordings from pyramidal neurons to examine the effects of SP on excitatory postsynaptic currents (EPSCs) mediated via AMPA receptors in the insular cortex (IC), which is involved in nociceptive information processing. First, EPSCs evoked by minimal electrical stimulation (eEPSCs) including stepwise EPSCs and failure events, were examined. SP dose-dependently suppressed mean eEPSC amplitude, partially due to an increase in the failure rate of eEPSCs. The SP-induced suppression of eEPSCs was accompanied by an increase in the paired-pulse ratio and was inhibited by the preapplication of SR140333, an NK1 receptor antagonist. [Sar9,Met(O2)11]-substance P, an NK1 receptor-selective agonist, mimicked the effects of SP on eEPSCs and decreased the frequency of miniature EPSCs (mEPSCs) without changing the average mEPSC amplitude. Considering that most NK1 receptors in the cerebral cortex are expressed in nitric oxide synthase (NOS)-positive GABAergic neurons, the SP-induced suppressive effect on EPSCs may be mediated by nitric oxide (NO) in this subtype of GABAergic neurons. NO imaging using the fluorescent probe DAX-J2 Red supports this hypothesis: SP increased the fluorescence intensity of DAX-J2 Red in some GABAergic neurons. Furthermore, both L-NAME, an NOS inhibitor, and PTIO, an NO scavenger, diminished the SP-induced suppression of eEPSCs. These results suggest that the activation of presynaptic NK1 receptors contributes to SP-induced eEPSC suppression by activating the NO synthesis pathway in GABAergic neurons. (246 words)

Activation of arginase II by asymmetric dimethylarginine and homocysteine in hypertensive rats induced by hypoxia: a new model of nitric oxide synthesis regulation in hypertensive processes?

In recent years, the increase in blood pressure at high altitudes has become an interesting topic among high-altitude researchers. In our animal studies using Wistar rats, we observed the existence of two rat populations that exhibit differential physiological responses during hypoxic exposure. These rats were classified as hypoxia-induced hypertensive rats and nonhypertensive rats. A decrease in nitric oxide levels was reported in different hypertension models associated with increased concentrations of asymmetric dimethylarginine (ADMA) and homocysteine, and we recently described an increase in arginase type II expression under hypoxia. ADMA and homocysteine decrease nitric oxide (NO) bioavailability; however, whether ADMA and homocysteine have a regulatory effect on arginase activity and therefore regulate another NO synthesis pathway is unknown. Therefore, the aim of this study was to measure basal ADMA and homocysteine levels in hypoxia-induced hypertensive rats and evaluate their effect on arginase II activity. Our results indicate that hypoxia-induced hypertensive rats presented lower nitric oxide concentrations than nonhypertensive rats, associated with higher concentrations of homocysteine and ADMA. Hypoxia-induced hypertensive rats also presented lower dimethylarginine dimethylaminohydrolase-2 and cystathionine β-synthase levels, which could explain the high ADMA and homocysteine levels. In addition, we observed that both homocysteine and ADMA had a significant effect on arginase II activation in the hypertensive rats. Therefore, we suggest that ADMA and homocysteine have dual regulatory effects on NO synthesis. The former has an inhibitory effect on eNOS, and the latter has a secondary activating effect on arginase II. We propose that arginase II is activated by AMDA and homocysteine in hypoxia-induced hypertensive rats.

Physical Exercise as a Multimodal Tool for COVID-19: Could It Be Used as a Preventive Strategy?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coronavirus disease 2019 (COVID-19) is a novel coronavirus not previously recognized in humans until late 2019. On 31 December 2019, a cluster of cases of pneumonia of unspecified etiology was reported to the World Health Organization in China. The availability of adequate SARS-CoV-2 drugs is also limited, and the efficacy and safety of these drugs for COVID-2019 pneumonia patients need to be assessed by further clinical trials. For these reasons, there is a need for other strategies against COVID-19 that are capable of prevention and treatment. Physical exercise has proven to be an effective therapy for most chronic diseases and microbial infections with preventive/therapeutic benefits, considering that exercise involves primary immunological mediators and/or anti-inflammatory properties. This review aimed to provide an insight into how the implementation of a physical exercise program against COVID-19 may be a useful complementary tool for prevention, which can also enhance recovery, improve quality of life, and provide immune protection against SARS-CoV-2 virus infection in the long term. In summary, physical exercise training exerts immunomodulatory effects, controls the viral gateway, modulates inflammation, stimulates nitric oxide synthesis pathways, and establishes control over oxidative stress.

Production and Characterization of Anti-Inflammatory Monascus Pigment Derivatives.

The prevention and treatment of chronic inflammation using food-derived compounds are desirable from the perspectives of marketing and safety. Monascus pigments, widely used as food additives, can be used as a chronic inflammation treatment. Orange Monascus pigments were produced by submerged fermentation in a 5 L bioreactor, and multiple orange Monascus pigment derivatives with anti-inflammatory activities were synthesized using aminophilic reaction. A total of 41 types of pigment derivatives were produced by incorporating amines and amino acids into the orange pigments. One derivative candidate that inhibited nitric oxide (NO) production in Raw 264.7 cells and exhibited low cell cytotoxicity was identified via in vitro assay. The 2-amino-4 picoline derivative inhibited NO production of 48.4%, and exhibited cell viability of 90.6%. Expression of inducible NO synthase, an important enzyme in the NO synthesis pathway, was suppressed by such a derivative in a dose-dependent manner. Therefore, this derivative has potential as a functional food colorant with anti-inflammatory effects.

Intraplatelet L-Arginine-Nitric Oxide Metabolic Pathway: From Discovery to Clinical Implications in Prevention and Treatment of Cardiovascular Disorders.

Despite the development of new drugs and other therapeutic strategies, cardiovascular disease (CVD) remains still the major cause of morbidity and mortality in the world population. A lot of research, performed mostly in the last three decades, revealed an important correlation between “classical” demographic and biochemical risk factors for CVD, (i.e., hypercholesterolemia, hyperhomocysteinemia, smoking, renal failure, aging, diabetes, and hypertension) with endothelial dysfunction associated directly with the nitric oxide deficiency. The discovery of nitric oxide and its recognition as an endothelial-derived relaxing factor was a breakthrough in understanding the pathophysiology and development of cardiovascular system disorders. The nitric oxide synthesis pathway and its regulation and association with cardiovascular risk factors were a common subject for research during the last decades. As nitric oxide synthase, especially its endothelial isoform, which plays a crucial role in the regulation of NO bioavailability, inhibiting its function results in the increase in the cardiovascular risk pattern. Among agents altering the production of nitric oxide, asymmetric dimethylarginine—the competitive inhibitor of NOS—appears to be the most important. In this review paper, we summarize the role of L-arginine-nitric oxide pathway in cardiovascular disorders with the focus on intraplatelet metabolism.

Inhibitors of nitric oxide synthase can reduce extracellular traps from neutrophils in asthmatic children in vitro.

This study aimed to explore the link between neutrophil extracellular traps (NETs) and childhood asthma, to investigate the ability of nitric oxide (NO) to induce NETs in asthmatic children and find inhibitors to reduce NETs in the NO synthesis pathway. A total of 49 children with mild persistent asthma were included in the study and 20 healthy children's blood samples were collected as healthy controls. Children with asthma were divided into symptomatic and asymptomatic groups according to the presence or absence of symptoms on the day of blood collection. Neutrophils in peripheral blood were isolated and plasma was preserved. NO donor (sodium nitroferricyanide(III) dehydrate [SNP]) could provide NO and proved by a fluorescent probe. A PicoGreen Kit was used to detect the NETs quantificationally. Fluorescence microscopy prepared to observe the main structures of NETs. We measured NETs components (extracellular free double-stranded DNA [dsDNA]) in healthy, symptomatic and asymptomatic groups' plasma samples, and we compared the ability of SNP with phosphate-buffered saline, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) to induce NETs. NO synthase (NOS) inhibitors were added to see the impact on NETs formation. Plasma was obtained from all blood samples of 69 children. The neutrophils of 40 asthmatic and 20 healthy children were successfully obtained, the recovery rate was over 95%, and the cell activity was over 80%. There was higher extracellular free dsDNA in the plasma of symptomatic group (n = 27) than asymptomatic group (n = 22) and healthy group (n = 20; P < .05). Studies on neutrophils from 40 children with asthma found that NO can be produced by adding SNP, PMA, and LPS. SNP could induce NETs with dose- and time-dependent. PMA (160 nM) had the strongest ability to induce NETs, LPS (200 ng/mL) followed, SNP (200 µM) was the weakest (P < .05), and the amount of NETs in the asthma group was significantly higher than that in the healthy group (P < .05). NOS inhibitors had the same blocking capacity for PMA- and LPS-induced NETs (P > .05), while NG-nitro- l-arginine methyl ester (500 µM) had the strongest inhibitory effect on SNP induction with time-dependent (P < .05). Inducible NOS was found in the NETs structure. Children with asthma had higher levels of NETs in peripheral blood, especially when they had asthma symptoms. We verified the ability of NO to induce NETs, and found neutrophils from asthmatic children can produce more NETs in vitro. NOS inhibitors blocked this process may provide new therapeutic targets for childhood asthma.

Nitric Oxide Remodels the Photosynthetic Apparatus upon S-Starvation in Chlamydomonas reinhardtii.

Many photosynthetic autotrophs have evolved responses that adjust their metabolism to limitations in nutrient availability. Here we report a detailed characterization of the remodeling of photosynthesis upon sulfur starvation under heterotrophy and photo-autotrophy in the green alga (Chlamydomonas reinhardtii). Photosynthetic inactivation under low light and darkness is achieved through specific degradation of Rubisco and cytochrome b 6 f and occurs only in the presence of reduced carbon in the medium. The process is likely regulated by nitric oxide (NO), which is produced 24 h after the onset of starvation, as detected with NO-sensitive fluorescence probes visualized by fluorescence microscopy. We provide pharmacological evidence that intracellular NO levels govern this degradation pathway: the addition of a NO scavenger decreases the rate of cytochrome b 6 f and Rubisco degradation, whereas NO donors accelerate the degradation. Based on our analysis of the relative contribution of the different NO synthesis pathways, we conclude that the NO2-dependent nitrate reductase-independent pathway is crucial for NO production under sulfur starvation. Our data argue for an active role for NO in the remodeling of thylakoid protein complexes upon sulfur starvation.

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

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

57 Novel roles for dietary nitrate in non ischaemic dilated cardiomyopathy and resistant hypertension

Introduction Non ischaemic dilated cardiomyopathy (NIDCM) and resistant hypertension (HTN) are common, chronic diseases. Blood nitrate/nitrite indicate nitric oxide (NO) bioavailability and levels are typically lower in NIDCM and HTN compared to controls indicating perturbed nitric oxide (NO) metabolism. In addition to the well-characterised L-arginine-NO synthase pathway, a second independent pathway for NO synthesis in vivo has been recently discovered whereby dietary nitrate is reduced to nitrite and consequently NO. Exploitation of this pathway represents a promising strategy for increasing NO bioavailability, with possible physiological benefits. Methods To assess effects in HTN, 19 eligible subjects wore an ambulatory blood pressure (BP) monitor (ABPM) for 24 h and fasting blood was taken before and after 14 d of nitrate-rich beetroot juice (BRJ). To assess effects in NIDCM, we recorded exercise capacity (incremental shuttle walk test) and BP before and 3 h after both BRJ and placebo in a randomised, double-blind, crossover trial among 11 subjects. There was 7 d between testing days with the alternate beverage. Results In the HTN study, 11 subjects had controlled blood pressure while 8 had uncontrolled blood pressure. There were similar increases in serum nitrate (p = 0.01) and nitrite (p = 0.001) in both controlled and uncontrolled hypertension. Further, there were no differences in either group regarding serum lipids (triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol). We observed very little chance in BP variables among the controlled hypertensives. In contrast, there were reductions in 24 h, daytime, and nighttime BP as well an increase in nocturnal BP dipping. The decreases in mean nighttime DBP and ambulatory arterial stiffness index (AASI) reached significance (p = 0.03 and p = 0.05 respectively). In the NIDCM trial, there were no significant differences in heart rate, arterial oxygen concentrations, breathlessness, leg fatigue or blood pressure at any time point with either beverage. However, NIDCM subjects walked significantly when took BRJ (+65 m) than when they took PL (-5 m) (p = 0.006). Conclusions Dietary nitrate has potential as a novel therapeutic, adjunct strategy to increase NO bioavailability in cardiovascular disease with potential physiological benefits. Our preliminary results require confirmation in larger trials. However, considering the low cost and safety profile of dietary nitrate containing foods and supplements, this concept appears promising as an adjunct therapeutic strategy for cardiovascular diseases.

Targeted arginine metabolomics: A rapid, simple UPLC-QToF-MSE based approach for assessing the involvement of arginine metabolism in human disease

BackgroundNitric oxide synthase (NOS) mediated conversion of arginine (ARG) to citrulline (CIT) is a key pathway for nitric oxide synthesis. ARG is also metabolised by alternate pathways to ornithine (ORN), homoarginine (HMA), NG-monomethyl-L-arginine (MMA), NG,NG-dimethyl-L-arginine (ADMA) and NG,NG′-dimethyl-L-arginine (SDMA), all of which have the capacity to alter NOS activity. Simultaneous assessment of these analytes, when assessing the impact of arginine metabolism in human disease states, is desirable. MethodsAnalytes (ARG, ADMA, SDMA, MMA, HMA, CIT and ORN) were isolated from human plasma by solvent extraction, evaporated and reconstituted. Ultra-performance liquid chromatography (UPLC) was performed on a 150mm×2.1mm T3 HSS column using a gradient mobile phase comprising ammonium formate (10mM, pH3.8) in methanol (1% to 63%). Analytes were detected by time-of-flight mass spectrometry (Q-ToF-MS) in positive ion mode with electrospray ionisation (ESI+). Data were collected using MSE. ResultsSolvent extraction provided high recovery (>95%). UPLC-QToF-MSE facilitated the separation and quantification of the 7 analytes in an analysis time of 6min. The approach has high sensitivity; LOQ range from 0.005μM (NMMA) to 0.25μM (ARG and ORN), and good precision; intra- and inter-day %RSD are <6% for all analytes. ConclusionsThis approach provides the capacity to quantify 7 key compounds involved in ARG metabolism in a small sample volume, with a short total analysis time. These characteristics make this approach ideal for undertaking a comprehensive characterisation of this pathway in large data sets (e.g. population studies).

Vasorelaxant effect of formononetin in the rat thoracic aorta and its mechanisms

The purpose of the present study was to investigate the effect of formononetin and the related mechanisms on isolated rat thoracic aorta. Formononetin concentration dependently relaxed aortic rings precontracted with norepinephrine (NE, 1 μM) or KCl (80 mM). Pretreatment with formononetin noncompetitively inhibited contractile responses of aortas to NE and KCl. The vasorelaxant effect of formononetin partially relied on intact endothelia, which was significantly attenuated by incubation with Nω-nitro-l-arginine methyl ester (100 μM). In endothelium-denuded rings, glibenclamide (10 μM) and tetraethylammonium (5 mM) showed slight reduction in the vasorelaxant effect of formononetin. Moreover, formononetin reduced NE-induced transient contraction in Ca2+-free solution and inhibited the vasocontraction induced by increasing external calcium in medium plus 80 mM KCl. Our results suggested that formononetin induced relaxation in rat aortic rings through an endothelium-dependent manner via nitric oxide synthesis pathway, and also involving an endothelium-independent vasodilatation by the blockade of Ca2+ channels. The opening of K+ channels might also be one of the mechanisms of formononetin-induced vasorelaxation.

Genetic variations in nitric oxide synthase and arginase influence exhaled nitric oxide levels in children

Exhaled nitric oxide (FeNO) is a biomarker of airway inflammation. In the nitric oxide (NO) synthesis pathway, nitric oxide synthases (encoded by NOS1, NOS2A, and NOS3) and arginases (encoded by ARG1 and ARG2) compete for L-arginine. Although FeNO levels are higher in children with asthma/allergy, influence of these conditions on the relationships between variations in these genes and FeNO remains unknown. The aims of the study were to evaluate the role of genetic variations in nitric oxide synthases and arginases on FeNO in children and to assess the influence of asthma and respiratory allergy on these genetic associations. Among children (6-11 years) who participated in the southern California Children's Health Study, variations in these five genetic loci were characterized by tagSNPs. FeNO was measured in two consecutive years (N = 2298 and 2515 in Years 1 and 2, respectively). Repeated measures analysis of variance was used to evaluate the associations between these genetic variants and FeNO. Sequence variations in the NOS2A and ARG2 loci were globally associated with FeNO (P = 0.0002 and 0.01, respectively). The ARG2 association was tagged by intronic variant rs3742879 with stronger association with FeNO in asthmatic children (P-interaction = 0.01). The association of a NOS2A promoter haplotype with FeNO varied significantly by rs3742879 genotypes and by asthma. Variants in the NO synthesis pathway genes jointly contribute to differences in FeNO concentrations. Some of these genetic influences were stronger in children with asthma. Further studies are required to confirm our findings.

Toward Metabolomic Signatures of Cardiovascular Disease

Small biochemicals are the end result of all the regulatory complexity present in a cell, tissue, or organism, including transcriptional regulation, translational regulation, and posttranslational modifications. Metabolic changes are thus the most proximal reporters of the body’s response to a disease process or drug therapy. In 1971, Robinson and coworkers1 conceived the core idea that information-rich data reflecting the functional status of a complex biological system resides in the quantitative and qualitative pattern of metabolites in body fluids. In the same year, Horning and Horning2 first used the term metabolic profiling to describe the output of a gas chromatogram from a patient sample. This new approach to the quantitative metabolic profiling of large numbers of small molecules in biofluids was ultimately termed “metabonomics” by Nicholson et al3 and “metabolomics” by others. Article see p 207 Two core technologies are used to perform metabolic profiling: nuclear magnetic resonance and tandem mass spectrometry (MS/MS), as previously reviewed in Circulation: Cardiovascular Genetics. 4 Nuclear magnetic resonance requires relatively little sample preparation and is nondestructive, allowing for subsequent structural analyses. However, the method tends to have low sensitivity and can detect only highly abundant analytes. Tandem mass spectrometry (MS/MS), coupled with liquid chromatography, on the other hand, has much higher sensitivity for small molecules and is also applicable to a wide range of biological fluids (including serum, plasma, and urine). Recent advances in MS technology now enable researchers to determine analyte masses with such high precision and accuracy that metabolites can be identified unambiguously even in complex fluids. These technologies can be used to characterize biological samples either in a targeted manner or in a pattern discovery manner. In the former, the investigator targets a predefined …

Single Nucleotide Polymorphism (SNP) Approach of Multiple Candidate Pathways Predicting the Risk of Acute / Chronic Graft-Versus-Host Disease or Transplant Outcomes Following Allogeneic Hematopoietic Stem Cell Transplantation: Potential Involvement of Nuclear Factor Kappa-B (NFKB), Platelet-Derived Growth Factor (PDGF) and Transforming Growth Factor-Beta (TGF-β) Pathway with Chronic Graft-Versus-Host Disease Graft-Versus-Host Disease.

Abstract 2221Poster Board II-198 Background:Acute graft-versus-host disease (GVHD) was known to be involved in the Th1 cytokine activation and alloreactive T-cell cytotoxicity, while the pathogenesis of chronic GVHD is yet revealed fully although in which Th2 cytokine activation or transforming growth factor (TGF) mediated pathway was suggested to be involved. The current study is a hypothesis generating study in order to identify potential predictive surrogate associated with the risk of acute or chronic GVHD in addition with transplant outcomes after allogeneic hematopoietic stem cell transplantation (HSCT). Methods:The current study was performed to identify genetic surrogates predicting the risk of acute / chronic GVHD, relapse free survival, non-relapse mortality and overall survival in 394 pairs transplanted at the Princess Margaret Hospital, Toronto, ON, Canada. In addition, the predictive markers for organ specific incidence of acute / chronic GVHD were also evaluated (i.e. for skin/liver/gut acute GVHD or skin, eye, oral, lung or liver chronic GVHD). Total of 261 single nucleotide polymorphisms (SNPs) in 56 genes were determined for donor/recipients' genotypes using MALDI-TOF based platform, involving in the pathways of 1) cytokines (i.e. IL1A, IL1B and its receptor, IL1R1, IL2 & IL2RA, IL4 & IL4R, IL6 & IL6R, IL8, IL10 & IL10RA, RB, IL12A/BandIL12RB1, IFNG & IFNGR1/2, TNFTI/II/II), 2) NFKB (NFKB1/2/A, NFKBIA/B, IKB, IKK1, IKBKB, RelB), 3) apoptosis (FAS, TRAIL & TRAILR1), 4) endothelium nitric oxide regulation (EDN1, NOS1/2A/3), 5) PDGF (PDGFB/C/D & PDGFRA/B), 6) TGF-β (TGFB1/2 & TGFBR1/2/3, TGFRB1), 7) Toll-like receptor (TLR4/5), 8) NOD2/CARD15 and 9) prostaglandin-endoperoxide synthase (PTGS1/2). The candidate genotypes have been selected by choosing the SNPs in non-synonymous SNPs in exon region with minor allele frequency of > 0.05 to 0.1. Results:Followings are the lists of recipients' and donors' genotypes with p-value<0.05 thus associating with clinical outcomes following allogeneic HSCT:Clinical outcomesRecipients' genotypeDonors' genotypeAcute GVHDFAS, EDN, TGFB1, TNFRI, NFKBIATNFRIChronic GVHD-IL1R, FCRG2AChronic GVHD, skinPDGFCNFKBA, PDGFCChronic GVHD, eyeIL6R, PDGFRTGFBR1Chronic GVHD, oralIL12RFCRG2AChronic GVHD, lungTGFB1, FAS, RelBIL4R, FAS, TGFB1, IL12B3Relapse free survivalIL6RNOD2/CARD15, IL6RNon-relapse mortalityIL2NOS2, TGFB2Overall survivalIL2NOS2, TGFB2, IL1BSteroid responsivenessIL4R, PDGFRIn summary, the risk of chronic GVHD was significantly associated with SNP of the genes involved in the pathway of NFKB, PDGF, TGF-β, and some of cytokines (esp. type II, IL6 & IL4), while that of acute GVHD associates with the genotypes in the pathway of TNF and apoptosis. In addition, survival after allogeneic transplantation was associated with the genotypes in NOS (nitric oxide synthase, endothelial nitric oxide synthesis pathway), IL-2 and TGF pathway. Conclusion:Because of complex nature of GVHD pathogenesis, multiple candidate pathway SNPs has been explored targeting SNPs in the pathway of cytokines, NFKB, apoptosis, endothelium nitric oxide regulation, NOD2/CARD15, PDGF, PTGS1/2, TGF-β and TLR. Different involvements were noted of TGF-β, PDGF or NFKB with chronic GVHD versus TNF and apoptosis-associated SNPs with acute GVHD. Further study will help us to reach more clear conclusion which genotype is the predictor of the risk of GVHD. Disclosures:No relevant conflicts of interest to declare.