Citrullination in the pathology of inflammatory and autoimmune disorders: recent advances and future perspectives.

Numerous post-translational modifications (PTMs) govern the collective metabolism of a cell through altering the structure and functions of proteins. The action of the most prevalent PTMs, encompassing phosphorylation, methylation, acylations, ubiquitination and glycosylation is well documented. A less explored protein PTM, conversion of peptidylarginine to citrulline, is the subject of this review. The process of citrullination is catalysed by peptidylarginine deiminases (PADs), a family of conserved enzymes expressed in a variety of human tissues. Accumulating evidence suggest that citrullination plays a significant role in regulating cellular metabolism and gene expression by affecting a multitude of pathways and modulating the chromatin status. Here, we will discuss the biochemical nature of arginine citrullination, the enzymatic machinery behind it and also provide information on the pathological consequences of citrullination in the development of inflammatory diseases (rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, periodontitis and COVID-19), cancer and thromboembolism. Finally, developments on inhibitors against protein citrullination and recent clinical trials providing a promising therapeutic approach to inflammatory disease by targeting citrullination are discussed.

Fine-Tuning Multi-Gene Clusters via Well-Characterized Gene Expression Regulatory Elements: Case Study of the Arginine Synthesis Pathway in C. glutamicum.

Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Corynebacterium glutamicum. Here, 17 core promoters and 29 RBSs in C. glutamicum were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs' strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to C. glutamicum, which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in C. glutamicum. Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanced, and the yield of a target metabolite can be further improved.

High-throughput enrichment of temperature-sensitive argininosuccinate synthetase for two-stage citrulline production in E. coli.

Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria.

Las concentraciones de glutamina y citrulina reflejan la síntesis del óxido nítrico en el sistema nervioso humano / Glutamine and citrulline concentrations reflect nitric oxide synthesis in the human nervous system

INTRODUCCIÓN: Aunque la citrulina es producida por la sintasa del óxido nítrico (NO) al activarse el receptor glutamatérgico NMDA, nitritos y nitratos (NOx) son considerados los mejores marcadores de síntesis del NO pues citrulina es también metabolizada por otras enzimas. En este estudio se realizó un análisis de correlaciones de concentraciones de citrulina y NOx en líquido cefalorraquídeo humano para evaluar la proporción en que la citrulina refleja la síntesis del NO y la neurotransmisión glutamatérgica. MÉTODOS: Se incluyeron pacientes con trastornos neurológicos agudos a los que se realizó punción lumbar (n = 240). Se determinó la concentración de NOx y aminoácidos por HPLC. RESULTADOS: NOx no fue diferente por la presencia de infección (p = 0,110) o inflamación (p = 0,349). La regresión múltiple mostró que NOx correlacionó con glutamina (r = -0,319, p < 0,001) y citrulina (r = 0,293, p = 0,005) pero no con el cociente citrulina/arginina (r = -0,160, p = 0,173). El ANCOVA confirmó que NOx está asociado con citrulina (F = 7,6, p = 0,007) pero no con el cociente citrulina/arginina (F = 2,2; p = 0,136), ni con la infección (F = 1,8; p = 0,173) o la inflamación (F = 1,4; p = 0,227). NOx no correlacionó con arginina ni con glutamato. CONCLUSIONES: Esto sugiere que citrulina refleja parte de la síntesis del NO a pesar de la contribución de otras vías metabólicas. Además, este estudio muestra que la glutamina es un modulador importante de la actividad de la NOS, y que arginina y glutamato no correlacionan con la concentración de NOx

INTRODUCTION: Although citrulline is produced by nitric oxide (NO) synthase upon activation of the NMDA glutamate receptor, nitrite and nitrate (NOx) concentration is considered the best marker of NO synthesis, as citrulline is also metabolised by other enzymes. This study analyses the correlation between human cerebrospinal fluid NOx and citrulline concentrations in order to determine the extent to which citrulline reflects NO synthesis and glutamatergic neurotransmission. METHODS: Participants were patients with acute neurological diseases undergoing lumbar puncture (n = 240). NOx and amino acid concentrations were determined by HPLC. RESULTS: NOx concentrations did not vary significantly where infection (p = 0,110) or inflammation (p = 0,349) were present. Multiple regression analysis showed that NOx concentration was correlated with glutamine (r = -0,319, p < 0,001) and citrulline concentrations (r = 0,293, p = 0,005) but not with the citrulline/arginine ratio (r = -0,160, p = 0,173). ANCOVA confirmed that NOx concentration was correlated with citrulline concentration (F = 7,6, p = 0,007) but not with the citrulline/arginine ratio (F = 2,2, p = 0,136), or presence of infection (F=1,8, p = 0,173) or inflammation (F = 1,4, p = 0,227). No association was found between NOx and arginine or glutamate concentrations. CONCLUSION: The results suggest that CSF citrulline concentration reflects NOx synthesis to some extent, despite the contribution of other metabolic pathways. In addition, this study shows that glutamine is an important modulator of NO synthase activity, and that arginine and glutamate are not correlated with NOx

Glutamine and citrulline concentrations reflect nitric oxide synthesis in the human nervous system. / Las concentraciones de glutamina y citrulina reflejan la síntesis del óxido nítrico en el sistema nervioso humano.

INTRODUCTION: Although citrulline is produced by nitric oxide (NO) synthase upon activation of the NMDA glutamate receptor, nitrite and nitrate (NOx) concentration is considered the best marker of NO synthesis, as citrulline is also metabolised by other enzymes. This study analyses the correlation between human cerebrospinal fluid NOx and citrulline concentrations in order to determine the extent to which citrulline reflects NO synthesis and glutamatergic neurotransmission. METHODS: Participants were patients with acute neurological diseases undergoing lumbar puncture (n=240). NOx and amino acid concentrations were determined by HPLC. RESULTS: NOx concentrations did not vary significantly where infection (p=0,110) or inflammation (p=0,349) were present. Multiple regression analysis showed that NOx concentration was correlated with glutamine (r=-0,319, p<0,001) and citrulline concentrations (r=0,293, p=0,005) but not with the citrulline/arginine ratio (r=-0,160, p=0,173). ANCOVA confirmed that NOx concentration was correlated with citrulline concentration (F=7,6, p=0,007) but not with the citrulline/arginine ratio (F=2,2, p=0,136), or presence of infection (F=1,8, p=0,173) or inflammation (F=1,4, p=0,227). No association was found between NOx and arginine or glutamate concentrations. CONCLUSION: The results suggest that CSF citrulline concentration reflects NOx synthesis to some extent, despite the contribution of other metabolic pathways. In addition, this study shows that glutamine is an important modulator of NO synthase activity, and that arginine and glutamate are not correlated with NOx.

Development of a System of High Ornithine and Citrulline Production by a Plant-Derived Lactic Acid Bacterium, Weissella confusa K-28.

As a bacterium used in industry for production of several amino acids, an endotoxin-free Corynebacterium (C.) glutamicum is well known. However, it is also true that the endotoxin-producing other Corynebacterium species is present. An aim of this study is to obtain a lactic acid bacterium (LAB) that produces ornithine and citrulline at high levels. We successfully isolated a strain, designated K-28, and identified it as Weissella (W.) confusa. The production of ornithine and citrulline by K-28 was 18 ± 1 and 10 ± 2 g/L, respectively, with a 100 ± 9% conversion rate when arginine was continuously fed into a jar fermenter. Although the ornithine high production using C. glutamicum is industrially present, the strains have been genetically modified. In that connection, the wild-type of C. glutamicum produces only 0.5 g/L ornithine, indicating that W. confusa K-28 is superior to C. glutamicum to use a probiotic microorganism. We confirmed that W. confusa K-28 harbors an arginine deiminase (ADI) gene cluster, wkaABDCR. The production of ornithine and the expression of these genes significantly decreased under the aerobic condition rather than anaerobic one. The expression level of the five genes did not differ with or without arginine, suggesting that the production of amino acids in the K-28 strain was not induced by exogenous arginine.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Systematized biosynthesis and catabolism regulate citrulline accumulation in watermelon.

Citrulline, a non-protein amino acid, is present in large amounts in watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai Cucurbitaceae) fruits. Amino acid profiling of various tissues of cv. Charleston Gray during plant development confirmed progressive accumulation of citrulline only in the fruit flesh and rind tissues. Citrulline content was positively correlated with precursor (ornithine) and by-product (arginine) amino acids during fruit ripening. Genetic variation in the partitioning of citrulline and related amino acids in the flesh and rind tissues was confirmed in a sub-set of watermelon cultivars. No correlation was established between morphological fruit traits (size and rind properties) and citrulline content. To understand the regulation of citrulline accumulation, we investigated the expression of genes associated with its biosynthesis and catabolism in flesh and rind tissues during fruit development. The expression of ornithine carbamoyltransferase (OTC) involved in the ultimate step of citrulline synthesis remained steady in both tissues. The expression of N-acetylornithine aminotransferase (N-AOA) involved in the production of N-acetylornithine and N-acetylornithine deacetylase (AOD-3) involved in ornithine synthesis coincided with increasing accumulation of citrulline in flesh and rind tissues during fruit development. Down-regulation N-acetylornithine-glutamate acetyltransferase (N-AOGA) suggests the subordinate role of the non-cyclic pathway in citrulline synthesis. Eccentricity between citrulline accumulation and expression of carbamoyl phosphate synthases (CPS-1, CPS-2) during fruit development suggest that the localized synthesis of carbamoyl phosphates may not be required for citrulline synthesis. Most genes involved in citrulline break-down (Argininosuccinate synthases - ASS-1, ASS-2, and ASS-3, Argininosuccinate lyases - ASL-1, Ornithine decarboxylase - ODC, Arginine decarboxylase - ADC) were consistently down-regulated during fruit development.

Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice.

Background: The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective: The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods: Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results: Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg-1 · h-1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA-1 · d-1; P < 0.001). Conclusions: Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

Dysbiotic Subgingival Microbial Communities in Periodontally Healthy Patients With Rheumatoid Arthritis.

OBJECTIVE: Studies that demonstrate an association between rheumatoid arthritis (RA) and dysbiotic oral microbiomes are often confounded by the presence of extensive periodontitis in these individuals. This study was undertaken to investigate the role of RA in modulating the periodontal microbiome by comparing periodontally healthy individuals with RA to those without RA. METHODS: Subgingival plaque was collected from periodontally healthy individuals (22 with RA and 19 without RA), and the 16S gene was sequenced on an Illumina MiSeq platform. Bacterial biodiversity and co-occurrence patterns were examined using the QIIME and PhyloToAST pipelines. RESULTS: The subgingival microbiomes differed significantly between patients with RA and controls based on both community membership and the abundance of lineages, with 41.9% of the community differing in abundance and 19% in membership. In contrast to the sparse and predominantly congeneric co-occurrence networks seen in controls, RA patients revealed a highly connected grid containing a large intergeneric hub anchored by known periodontal pathogens. Predictive metagenomic analysis (PICRUSt) demonstrated that arachidonic acid and ester lipid metabolism pathways might partly explain the robustness of this clustering. As expected from a periodontally healthy cohort, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were not significantly different between groups; however, Cryptobacterium curtum, another organism capable of producing large amounts of citrulline, emerged as a robust discriminant of the microbiome in individuals with RA. CONCLUSION: Our data demonstrate that the oral microbiome in RA is enriched for inflammophilic and citrulline-producing organisms, which may play a role in the production of autoantigenic citrullinated peptides in RA.

Hypoxia induces production of citrullinated proteins in human fibroblast-like synoviocytes through regulating HIF1α.

Hypoxia is a prominent microenvironment feature in a range of disorders including cancer, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel disease (IBD), infection and obesity. Hypoxia promotes biological functions of fibroblast-like synoviocytes via regulating hypoxia-inducible factor 1α (HIF1α). Dysregulated protein citrullination in RA drives the production of antibodies to citrullinated proteins, a highly specific biomarker of RA. However, the mechanisms promoting citrullination in RA are not yet fully elucidated. In this study, we investigated whether pathophysiological hypoxia as found in the rheumatoid synovium modulates the citrullination in human fibroblast-like synoviocytes (HFLS). Here, we found that peptidylarginine deiminase 2 (PAD2) and citrullinated proteins were increased in HFLS after exposure to hypoxia. Moreover, knocking down HIF1α by HIF1α siRNA ameliorated the expression of PAD2 and citrullinated proteins. Collectively, this study provides a new mechanism involved in generating citrullinated proteins: hypoxia promotes citrullination and PAD production in HFLS. Concurrently, we also proposed a novel hypoxia involved mechanism in RA pathogenesis. This study deepens our understanding of the role of hypoxia in the pathogenesis of RA and provides a potential therapeutic strategy for RA.

8-year retrospective analysis of intravenous arginine therapy for acute metabolic strokes in pediatric mitochondrial disease.

BACKGROUND: Intravenous (IV) arginine has been reported to ameliorate acute metabolic stroke symptoms in adult patients with Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes (MELAS) syndrome, where its therapeutic benefit is postulated to result from arginine acting as a nitric oxide donor to reverse vasospasm. Further, reduced plasma arginine may occur in mitochondrial disease since the biosynthesis of arginine's precursor, citrulline, requires ATP. Metabolic strokes occur across a wide array of primary mitochondrial diseases having diverse molecular etiologies that are likely to share similar pathophysiologic mechanisms. Therefore, IV arginine has been increasingly used for the acute clinical treatment of metabolic stroke across a broad mitochondrial disease population. METHODS: We performed retrospective analysis of a large cohort of subjects who were under 18 years of age at IRB #08-6177 study enrollment and had molecularly-confirmed primary mitochondrial disease (n = 71, excluding the common MELAS m.3243A>G mutation). 9 unrelated subjects in this cohort received acute arginine IV treatment for one or more stroke-like episodes (n = 17 total episodes) between 2009 and 2016 at the Children's Hospital of Philadelphia. Retrospectively reviewed data included subject genotype, clinical symptoms, age, arginine dosing, neuroimaging (if performed), prophylactic therapies, and adverse events. RESULTS: Genetic etiologies of subjects who presented with acute metabolic strokes included 4 mitochondrial DNA (mtDNA) pathogenic point mutations, 1 mtDNA deletion, and 4 nuclear gene disorders. Subject age ranged from 19 months to 23 years at the time of any metabolic stroke episode (median, 8 years). 3 subjects had recurrent stroke episodes. 70% of subjects were on prophylactic arginine or citrulline therapy at the time of a stroke-like episode. IV arginine was initiated on initial presentation in 65% of cases. IV arginine was given for 1-7 days (median, 1 day). A positive clinical response to IV arginine occurred in 47% of stroke-like episodes; an additional 6% of episodes showed clinical benefit from multiple simultaneous treatments that included arginine, confounding sole interpretation of arginine effect. All IV arginine-responsive stroke-like episodes (n = 8) received treatment immediately on presentation (p = .003). Interestingly, the presence of unilateral symptoms strongly predicted arginine response (p = .02, Chi-Square); however, almost all of these cases immediately received IV arginine, confounding interpretation of causality direction. Suggestive trends toward increased IV arginine response were seen in subjects with mtDNA relative to nDNA mutations and in older pediatric subjects, although statistical significance was not reached possibly due to small sample size. No adverse events, including hypotensive episodes, from IV arginine therapy were reported. CONCLUSIONS: Single-center retrospective analysis suggests that IV arginine therapy yields significant therapeutic benefit with little risk in pediatric mitochondrial disease stroke subjects across a wide range of genetic etiologies beyond classical MELAS. Acute hemiplegic stroke, in particular, was highly responsive to IV arginine treatment. Prospective studies with consistent arginine dosing, and pre- and post-neuroimaging, will further inform the clinical utility of IV arginine therapy for acute metabolic stroke in pediatric mitochondrial disease.

[Production of L-citrulline by a recombinant Corynebacterium crenatum SYPA 5-5 whole-cell biocatalyst].

Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 µmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.

Citrulline metabolism in plants.

Citrulline was chemically isolated more than 100 years ago and is ubiquitous in animals, plants, bacteria, and fungi. Most of the research on plant citrulline metabolism and transport has been carried out in Arabidopsis thaliana and the Cucurbitaceae family, particularly in watermelon which accumulates this non-proteinogenic amino acid to very high levels. Industrially, citrulline is produced via specially optimized microbial strains; however, the amounts present in watermelon render it an economically viable source providing that other high-value compounds can be co-extracted. In this review, we provide an overview of our current understanding of citrulline biosynthesis, transport, and catabolism in plants additionally pointing out significant gaps in our knowledge which need to be closed by future experimentation. This includes the identification of further potential enzymes of citrulline metabolism as well as obtaining a far better spatial resolution of both sub-cellular and long-distance partitioning of citrulline. We further discuss what is known concerning the biological function of citrulline in plants paying particular attention to the proposed roles in scavenging of excess NH4+ and as a compatible solute.

Plasma Glutamine Is a Minor Precursor for the Synthesis of Citrulline: A Multispecies Study.

Background: Glutamine is considered the main precursor for citrulline synthesis in many species, including humans. The transfer of 15N from 2-[15N]-glutamine to citrulline has been used as evidence for this precursor-product relation. However, work in mice has shown that nitrogen and carbon tracers follow different moieties of glutamine and that glutamine contribution to the synthesis of citrulline is minor. It is unclear whether this small contribution of glutamine is also true in other species.Objective: The objective of the present work was to determine the contribution of glutamine to citrulline production by using nitrogen and carbon skeleton tracers in multiple species.Methods: Humans (n = 4), pigs (n = 5), rats (n = 6), and mice (n = 5) were infused with l-2-[15N]- and l-[2H5]-glutamine and l-5,5-[2H2]-citrulline. The contribution of glutamine to citrulline synthesis was calculated by using different ions and fragments: glutamine M+1 to citrulline M+1, 2-[15N]-glutamine to 2-[15N]-citrulline, and [2H5]-glutamine to [2H5]-citrulline.Results: Species-specific differences in glutamine and citrulline fluxes were found (P < 0.001), with rats having the largest fluxes, followed by mice, pigs, and humans (all P < 0.05). The contribution of glutamine to citrulline as estimated by using glutamine M+1 to citrulline M+1 ranged from 88% in humans to 46% in pigs. However, the use of 2-[15N]-glutamine and 2-[15N]-citrulline as precursor and product yielded values of 48% in humans and 28% in pigs. Furthermore, the use of [2H5]-glutamine to [2H5]-citrulline yielded lower values (P < 0.001), resulting in a contribution of glutamine to the synthesis of citrulline of ∼10% in humans and 3% in pigs.Conclusions: The recycling of the [15N]-glutamine label overestimates the contribution of glutamine to citrulline synthesis compared with a tracer that follows the carbon skeleton of glutamine. Glutamine is a minor precursor for the synthesis of citrulline in humans, pigs, rats, and mice.

Determine exogenous human DDAH2 gene function in rabbit bone marrow-derived endothelial progenitor cells in vitro.

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2-EPCs). Three days after gene transfer, positive transfected-EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium-like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk-1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.

Interrelationships between glutamine and citrulline metabolism.

PURPOSE OF REVIEW: This article analyzes the contribution of glutamine to the synthesis of citrulline and reviews the evidence that glutamine supplementation increases citrulline production. RECENT FINDINGS: Glutamine supplementation has been proposed in the treatment of critically ill patients; however, a recent large multicenter randomized controlled trial resulted in increased mortality in the glutamine-supplemented group. Within this context, defining the contribution of glutamine to the production of citrulline, and thus to de-novo arginine synthesis, has become a pressing issue. SUMMARY: The beneficial effects of glutamine supplementation may be partially mediated by the effects of glutamine on citrulline synthesis by the gut and the de-novo synthesis of arginine by the kidney and other tissues. Although there is no strong evidence to support that glutamine is a major precursor for citrulline synthesis in humans, glutamine has the potential to increase overall gut function and in this way increase citrulline production.

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide.

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of ∼2.4·10(-5) and ∼7.9·10(-5) m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ∼1.5·10(-5) m at pH 6.0 and increased to ∼8.3·10(-5) m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of ∼1.9·10(-5) m in the presence of Arg and BH4 but exhibited much higher IC50 values (∼1.0-6.1·10(-4) m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 ∼ 1.3-2.0·10(-5) m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.

Dietary arginine requirements for growth are dependent on the rate of citrulline production in mice.

BACKGROUND: In many species, including humans, arginine is considered a semiessential amino acid because under certain conditions endogenous synthesis cannot meet its demand. The requirements of arginine for growth in mice are ill defined and seem to vary depending on the genetic background of the mice. OBJECTIVE: The objective of this study was to determine the metabolic and molecular basis for the requirement of arginine in 2 mouse strains. METHODS: Institute of Cancer Research (ICR) and C57BL/6 (BL6) male mice were fed arginine-free or arginine-sufficient diets (Expt. 1) or 1 of 7 diets with increasing arginine concentration (from 0- to 8-g/kg diet, Expt. 2) between day 24 and 42 of life to determine the arginine requirements for growth. Citrulline production and "de novo" arginine synthesis were measured with use of stable isotopes, and arginine requirements were determined by breakpoint analysis and enzyme expression by reverse transcriptase-polymerase chain reaction. RESULTS: In Expt. 1, ICR mice grew at the same rate regardless of the arginine concentration of the diet (mean ± SE: 0.66 ± 0.04 g/d, P = 0.80), but BL6 mice had a reduced growth rate when fed the arginine-free diet (0.25 ± 0.02 g/d, P < 0.001) compared to the 8-g arginine/kg diet (0.46 ± 0.03 g/d). ICR mice showed at least a 2-fold greater expression (P < 0.001) of ornithine transcarbamylase (OTC) than BL6 mice, which translated into a greater rate of citrulline (25%) and arginine synthesis (49%, P < 0.002). In Expt. 2, breakpoint analysis showed that the requirement for growth of BL6 mice was met with 2.32 ± 0.39 g arginine/kg diet; for ICR mice, however, no breakpoint was found. CONCLUSION: Our data indicate that a reduced expression of OTC in BL6 mice translates into a reduced production of citrulline and arginine compared with ICR mice, which results in a dietary arginine requirement for growth in BL6 mice, but not in ICR mice.

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis.

INTRODUCTION: Smoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4). METHODS: Lung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test. RESULTS: Within the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase. CONCLUSIONS: We have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.