Pyrrolidine dithiocarbamate in combination with L-N-monomethyl arginine alleviates Staphylococcus aureus infection via regulation of CXCL8/CXCR1 axis in peritoneal macrophages in vitro.

The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, H2O2, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.

Methylarginine efflux in nutrient-deprived yeast mitigates disruption of nitric oxide synthesis.

Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell signaling. Intracellular free methylarginines contribute to cellular stress through their interaction with nitric oxide synthase (NOS). The arginine-dependent production of nitric oxide (NO), which is strongly inhibited by methylarginines, serves as a protective small molecule against oxidative stress in eukaryotic cells. NO signaling is highly conserved between higher and lower eukaryotes, although a canonical NOS homologue has yet to be identified in yeast. Since stress signaling pathways are well conserved among eukaryotes, yeast is an ideal model organism to study the implications of PRMTs and methylarginines during stress. We sought to explore the roles and fates of methylarginines in Saccharomyces cerevisiae. We starved methyltransferase-, autophagy-, and permease-related yeast knockouts by incubating them in water and monitored methylarginine production. We found that under starvation, methylarginines are expelled from yeast cells. We found that autophagy-deficient cells have an impaired ability to efflux methylarginines, which suggests that methylarginine-containing proteins are degraded via autophagy. For the first time, we determine that yeast take up methylarginines less readily than arginine, and we show that methylarginines impact yeast NO production. This study reveals that yeast circumvent a potential methylarginine toxicity by expelling them after autophagic degradation of arginine-modified proteins.

[Effect of astragaloside â £ on angiotensin â ¡-induced inflammatory response of vascular endothelial cells and mechanism].

This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin Ⅱ(Ang Ⅱ), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-Ⅳ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang Ⅱ stimulation. The inhibitory effect of AS-Ⅳ on Ang Ⅱ-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang Ⅱ-stimulated endothelial cells. AS-Ⅳ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-Ⅳ-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang Ⅱ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang Ⅱ-induced inflammatory response was inhibited by pretreatment with AS-Ⅳ(P<0.05). In addition, the inhibitory effect of AS-Ⅳ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-Ⅳ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-Ⅳ-mediated anti-inflammatory actions in endothelial cells exposed to Ang Ⅱ. The results indicate that AS-Ⅳ attenuates endothelial cell-mediated inflammatory response induced by Ang Ⅱ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.

Endothelial cell-specific loss of eNOS differentially affects endothelial function.

The endothelium maintains and regulates vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating Nitric Oxide (NO) availability. Endothelial nitric oxide synthase (eNOS) is one of three NOS isoforms that catalyses the synthesis of NO to regulate endothelial function. However, eNOS's role in the regulation of endothelial function, such as cell proliferation and migration remain unclear. To gain a better understanding, we genetically knocked down eNOS in cultured endothelial cells using sieNOS and evaluated cell proliferation, migration and also tube forming potential in vitro. To our surprise, loss of eNOS significantly induced endothelial cell proliferation, which was associated with significant downregulation of both cell cycle inhibitor p21 and cell proliferation antigen Ki-67. Knockdown of eNOS induced cell migration but inhibited formation of tube-like structures in vitro. Mechanistically, loss of eNOS was associated with activation of MAPK/ERK and inhibition of PI3-K/AKT signaling pathway. On the contrary, pharmacologic inhibition of eNOS by inhibitors L-NAME or L-NMMA, inhibited cell proliferation. Genetic and pharmacologic inhibition of eNOS, both promoted endothelial cell migration but inhibited tube-forming potential. Our findings confirm that eNOS regulate endothelial function by inversely controlling endothelial cell proliferation and migration, and by directly regulating its tube-forming potential. Differential results obtained following pharmacologic versus genetic inhibition of eNOS indicates a more complex mechanism behind eNOS regulation and activity in endothelial cells, warranting further investigation.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

BACKGROUND: The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC. AIM: To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODS: Male rats (n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTS: The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions. CONCLUSION: In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Anti-Acne Vulgaris Effects of Pedunculagin from the Leaves of Quercus mongolica by Anti-Inflammatory Activity and 5α-Reductase Inhibition.

Quercus mongolica (QM)-a member of the Fagaceae family-has been used as traditional medicine in Korea, China and Mongolia as a treatment for inflammation of oral, genital or anal mucosa and for external inflammation of skin. To treat acne vulgaris (AV), we evaluated the inhibition of inflammatory cytokines (IL-6 and IL-8) of QM leaf extract (QML) and its main compound, pedunculagin (PD) in vitro and 5α-reductase inhibitory activity by western blotting. As results, QML and PD showed potent NO production inhibitory activity compared with the positive control (PC), NG-monomethyl-L-arginine (L-NMMA). QML and PD was also showed the decreases of IL-6 and IL-8 compared with the PC, EGCG and exhibited potent 5α-reductase type 1 inhibitory activities compared with the PC, dutasteride.

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Total methylated arginine load as a risk parameter in subjects with masked hypertension.

Asymmetric dimethylarginine, symmetric dimethylarginine, and L-monomethylarginine are originated from the subsequent proteolytic catalysis of methylated arginine residues on different proteins and inhibit the endogenous nitric oxide generation. The changes in total methylarginine load (Asymmetric dimethylarginine plus symmetric dimethylarginine plus L-monomethylarginine) may contribute to hypertension. The aim of this study was to determine serum methylarginine concentrations in patients with masked hypertension and determine the association between these biomarkers and blood pressure measurements. Control group, masked hypertension and hypertension groups consisted of 40 subjects (11 males, 28 females, mean age 48.6 ± 13.1), 28 subjects (14 males, 14 females, mean age 50.9 ± 11.0) and 36 subjects (15 males, 21 females, mean age 54.4 ± 12.3 years), respectively (P= 0.149). Serum total methylarginine load was significantly higher in hypertension group (0.63 ± 0.23) compared to masked hypertension (0.49 ± 0.16) and control groups (0.38 ± 0.13) (P= 0.008 and P< 0.001). While there was no statistically significant difference between healthy control groups [0.147 (0.03-0.29)] and masked hypertension patients [0.144 (0.05-0.42)] for serum symmetric dimethylarginine levels (P= 0.970), it was markedly elevated in hypertension group [0.25 (0.07-0.54)] compared to masked hypertension group [0.14 (0.05-0.42)] (P= 0.001). Serum total methylarginine load was positively correlated with night-time SBP (r = 0.214, P= 0.029). Serum methylarginine levels might be a useful marker for determining the courses of clinical hypertension.

Normal increases in insulin-stimulated glucose uptake after ex vivo contraction in neuronal nitric oxide synthase mu (nNOSµ) knockout mice.

Nitric oxide (NO) is involved in skeletal muscle glucose uptake during exercise and also in the increase in insulin sensitivity after exercise. Given that neuronal nitric oxide synthase (NOS) isoform mu (nNOSµ) is a major isoform of NOS in skeletal muscle, we examined if the increase in skeletal muscle insulin-stimulated glucose uptake 3.5 h following ex vivo contraction of extensor digitorum longus (EDL) is reduced in muscles from nNOSµ+/- and nNOSµ-/- mice compared with nNOSµ+/+ mice. 3.5 h post-contraction/basal, muscles were exposed to saline or insulin (120µU/ml) with or without the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) during the last 30 min and glucose uptake was determined by radioactive tracers. Skeletal muscle insulin-stimulated glucose uptake from nNOSµ+/+, nNOSµ+/-, and nNOSµ-/- mice increased approximately twofold 3.5 h following ex vivo contraction when compared to rest. L-NMMA significantly attenuated this increase in muscle insulin-stimulated glucose uptake by around 50%, irrespective of genotype. Low levels of NOS activity were detected in muscles from nNOSµ-/- mice. In conclusion, NO mediates increases in mouse skeletal muscle insulin response following ex vivo contraction independently of nNOSµ.

Asymmetric dimethylarginine (ADMA) as an important risk factor for the increased cardiovascular diseases and heart failure in chronic kidney disease.

Patients with chronic kidney disease have an increased cardiovascular morbidity and mortality. It has been recognized that the traditional cardiovascular risk factors could only partially explain the increased cardiovascular morbidity and mortality in patients with chronic kidney disease. Asymmetric dimethylarginine (ADMA) and N-monomethy l-arginine (L-NMMA) are endogenous inhibitors of nitric oxide synthases that attenuate nitric oxide production and enhance reactive oxidative specie generation. Increased plasma ADMA and/or L-NMMA are strong and independent risk factor for chronic kidney disease, and various cardiovascular diseases such as hypertension, coronary artery disease, atherosclerosis, diabetes, and heart failure. Both ADMA and L-NMMA are also eliminated from the body through either degradation by dimethylarginine dimethylaminohydrolase-1 (DDAH1) or urine excretion. This short review will exam the literature of ADMA and L-NMMA degradation and urine excretion, and the role of chronic kidney diseases in ADMA and L-NMMA accumulation and the increased cardiovascular disease risk. Based on all available data, it appears that the increased cardiovascular morbidity in chronic kidney disease may relate to the dramatic increase of systemic ADMA and L-NMMA after kidney failure.

Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways.

RATIONALE: Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known. OBJECTIVE: Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity. METHODS AND MEASUREMENTS: 34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24-72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay. MAIN RESULTS: Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis. CONCLUSIONS: In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.

DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1.

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize NG ,NG -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including NG -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

ORF7 from Amycolatopsis orientalis catalyzes decarboxylation of Nδ-methylarginine and amine oxidation of arginine: Biosynthetic implications.

A key step in the biosynthesis of the polyene polyketide ECO-0501 by Amycolatopsis orientalis ATCC 43491 is thought to involve oxidative decarboxylation of arginine or Nδ-methylarginine to the corresponding primary amide. This reaction is the centerpiece of a recently identified biosynthetic cassette that generates 4-guanidinobutyryl thioesters to serve as starter units for polyketide synthesis. We examined the reaction of ORF7, the predicted ECO-0501 biosynthetic decarboxylase, with arginine, and saw no evidence of decarboxylation. Instead, we observed exclusive amine oxidation to generate 2-oxoarginine, with a kcat/KM,Arg of 5.6×106M-1s-1, typical of values measured for physiological amino acid decarboxylases. In contrast, when ORF7 was incubated with Nδ-methylarginine, we observed exclusive decarboxylation to generate 4-(N1-methylguanidino)butyramide. These differing reactive pathways provide insight into the biosyntheses of guanidinobutyryl-derived polyketides and demonstrate the biosynthetic versatility of arginine-processing decarboxylases. In addition, it suggests that ORF7 may be an incisive model system for dissecting the determinants of flavoprotein-catalyzed oxidase and monooxygenase modes of reactivity.

Effect of asymmetric dimethylarginine (ADMA) on heart failure development.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases that limits nitric oxide bioavailability and can increase production of NOS derived reactive oxidative species. Increased plasma ADMA is a one of the strongest predictors of mortality in patients who have had a myocardial infarction or suffer from chronic left heart failure, and is also an independent risk factor for several other conditions that contribute to heart failure development, including hypertension, coronary artery disease/atherosclerosis, diabetes, and renal dysfunction. The enzyme responsible for ADMA degradation is dimethylarginine dimethylaminohydrolase-1 (DDAH1). DDAH1 plays an important role in maintaining nitric oxide bioavailability and preserving cardiovascular function in the failing heart. Here, we examine mechanisms of abnormal NO production in heart failure, with particular focus on the role of ADMA and DDAH1.

Lymphocyte-mediated macrophage apoptosis during IL-12 stimulation.

In contrast to the well known immunostimulatory roles of IL-12, little has been known about its immunosuppressive roles. In the present study, IL-12-activated lymphocyte-mediated macrophage apoptosis was investigated by employing murine lymphocyte/macrophage cocultures. IL-12-activated lymphocytes and their culture supernatants induced an inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis in macrophages. The NO synthesis was markedly inhibited by blocking antibodies to IFN-γ and TNF-α, suggesting the key role of these lymphocyte cytokines in mediating the NO synthesis. The endogenously produced NO inhibited macrophage proliferation, and induced apoptosis in concordance with the accumulation of p53, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and DR5, and the activation of caspase-3, processes that were inhibited by N(G)-monomethyl-l-arginine, aminoguanidine (NO synthase inhibitors) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (an NO scavenger). These results were further supported by the findings obtained from the experiments employing IFN-γ-knockout and iNOS-knockout mice. Our study demonstrated a novel, non-contact-dependent mechanism of macrophage suppression by IL-12-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages due to endogenous NO synthesis induced by cytokines secreted from IL-12-activated lymphocytes.

Analysis of the proteome of Saccharomyces cerevisiae for methylarginine.

Arginine methylation is a post-translational modification that has been implicated in a plethora of cellular processes. In the present manuscript, using two antimethylarginine antibodies and combinatorial deletion mutants of arginine methyltransferases, we found evidence of widespread arginine methylation in the Saccharomyces cerevisiae proteome. Immunoprecipitation was used for enrichment of methylarginine-containing proteins, which were identified via tandem mass spectrometry. From this, we identified a total of 90 proteins, of which 5 were previously known to be methylated. The proteins identified were involved in known methylarginine-associated biological functions such as RNA processing, nuclear transport, carbohydrate metabolic process, GMP biosynthetic process and protein folding. Through in vivo methylation by the incorporation of [3H]-methyl groups, we validated the methylation of 7 proteins (Ded1, Imd4, Lhp1, Nop1, Cdc11, Gus1, Pob3). By LC-MS/MS, we then confirmed a total of 15 novel methylarginine sites on 5 proteins (Ded1, Lhp1, Nop1, Pab1, and Ugp1). By examination of methylation on proteins from the triple knockout of methyltransferases Hmt1, Hsl7, Rmt2, we present evidence for the existence of additional unidentified arginine methyltransferases in the Saccharomyces cerevisiae proteome.

Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats.

BACKGROUND AND AIM: The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. METHODS: Cirrhosis was induced in rats by CCl(4) inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-N(G)-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). RESULTS: The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. CONCLUSIONS: The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

Endogenous nitric oxide synthase inhibitors in the biology of disease: markers, mediators, and regulators?

The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring, and modulating circulating asymmetric dimethylarginine alone is inadequate. This review outlines the basic biochemistry and physiology of endogenous methylarginines, examines both the experimental and observational evidence for a role in disease pathogenesis, and examines the potential for therapeutic regulation of these molecules.

The temporal effect of a wild blueberry (Vaccinium angustifolium)-enriched diet on vasomotor tone in the Sprague-Dawley rat.

BACKGROUND AND AIMS: We have previously reported that wild blueberry (Vaccinium angustifolium)-enriched diets (WB) attenuate aortic adrenergic response through endothelial-mediated pathways. The duration of dietary intervention necessary to induce the positive changes on vasomotor tone has not been studied to date. Thus, our objective was to investigate the temporal effect of WB consumption on vascular function and reactivity in Sprague-Dawley (SD) rat aorta after 4 and 7 weeks of dietary treatment. METHODS AND RESULTS: Forty male SD rats were randomly assigned to a control (AIN-93) (C) or a WB diet for 4 or 7 weeks. Vascular ring studies were conducted in 3-mm isolated rat aortic rings to investigate vasoconstriction induced by six doses of the α(1)-adrenergic agonist, L-phenylephrine (Phe, 10(-8)-3×10(-6) M) alone or in the presence of the NOS inhibitor, L-N(G)-monomethyl-arginine (L-NMMA, 10(-4)M). The maximum force of contraction (F(max)) and vessel sensitivity (pD(2)) were determined. Analysis of variance revealed no significant differences on F(max) after 4 weeks of the WB diet but only a significant increase in pD(2) in the absence of L-NMMA. Seven week WB consumption significantly attenuated contraction in response to L-Phe and resulted in lower pD(2). Inhibition of NOS induced a significant increase in the constrictor response in both diet groups at both time periods, with the WB group fed for 7 weeks having the greater response. CONCLUSION: Thus wild blueberries incorporated into the diet at 8% w/w positively affect vascular smooth muscle contractility and sensitivity but these effects are evident only after 7 weeks of WB consumption.

In vivo nitric oxide suppression of lipolysis in subcutaneous abdominal adipose tissue is greater in obese than lean women.

Mounting evidence suggests there is a reduced mobilization of stored fat in obese compared to lean women. It has been suggested that this decreased lipid mobilization may lead to, or perpetuate, the obese state; however, there may be a beneficial effect of reduced lipolysis, either by allowing for a sink of excess fatty acids, or by limiting a potentially harmful rise in interstitial and circulating fatty acid concentration. Nitric oxide (NO) may be responsible for a portion of the reduced in vivo rates of lipolysis in obese women because NO reduces adipose tissue lipolysis and adipose tissue nitric oxide synthase (NOS) mRNA is higher in obese than lean individuals. The purpose of this study was to determine if the inhibition of NOS by L-N(g)-monomethyl-L-arginine (L-NMMA) in the absence and presence of lipolytic stimulation would result in a larger increase in lipolytic rate in obese (OB) than lean (LN) women. Microdialysis probes were inserted into the subcutaneous abdominal adipose tissue of seven obese and six lean women to monitor lipolysis. Dialysate glycerol concentration increased in response to L-NMMA in OB (basal 125 ± 26 µmol/l; L-NMMA 225 ± 35 µmol/l) to a greater extent than in LN (basal 70 ± 18 µmol/l; L-NMMA 84 ± 20 µmol/l) women (P < 0.05). Dialysate glycerol increased to a similar extent in OB and LN in response to adrenergic stimulation by isoprenaline or norepinephrine in the presence of L-NMMA. The differential glycerol responses to L-NMMA between obese and lean could not be explained by differential blood flow responses. It can be concluded that NO suppresses basal lipolysis in obese women to a greater extent than in lean women.