Arginase Gene Research Articles

Exploration of immunomodulatory and protective effect of Withania somnifera on trace metal oxide (zinc oxide nanoparticles) induced toxicity in Balb/c mice.

The current study was undertaken to investigate the immunomodulatory and protective effects of Withania somnifera (WS) extract and Withaferin A (WA) supplementation on zinc oxide nanoparticles mediated toxicity in Balb/c mice. The animals were exposed to ZnO NPs along with WS and WA for 28 days and various parameters like body weight, organ coefficient, cytotoxicity, nitric oxide (NO), total serum protein, phagocytosis, and the gene expression levels of TLR6 and ARG genes were determined. In vivo study showed that, dose-dependent reduction in phagocytosis, an increase in the levels of NO production along with up-regulation of TLR6, arginase gene was significant (P < 0.05) when ZnO NPs were given. However toxicity of ZnO NP was reduced in presence of WS and WA with decreased TLR6 over expression and restoration of phagocytic activities. Our results provided a valuable insight into the underlying mechanism for the protective effects of WS. Mechanism of toxicity induced by Zinc oxide nanoparticles ZnO NPs and immunomodulatory protective effects of Withania somnifera extract (WS) and Withaferin A (WA), in Balb/c mice modal of peritoneal macrophages. Red arrows: effect of ZnO NPs independently leads to ROS production which attenuated the phagocytosis of yeast by macrophages through, up-regulation of TLR6 and down-regulation of arginase gene expression. Green arrows: co-treatment, Impact of Withania somnifera extract with zinc oxide nanoparticles (WS + ZnO NPs), Withaferin A along with zinc oxide nanoparticles (WA + ZnO NPs)-enhance phagocytic activity by counteracting mechanism of ZnO NPs toxicity. Black arrows: increasing or decreasing effects. Per oral (P.O).

Spermidine-Regulated Biosynthesis of Heat-Stable Antifungal Factor (HSAF) in Lysobacter enzymogenes OH11.

Heat-Stable Antifungal Factor (HSAF) and its analogs are antifungal natural products produced by the biocontrol agent Lysobacter enzymogenes. The production of HSAF is greatly influenced by environmental stimuli and nutrients, but the underlying molecular mechanism is mostly unclear. Here, we found that HSAF production in L. enzymogenes OH11 is strictly controlled by spermidine, which is the most prevalent triamine in bacteria. When added into OH11 cultures, spermidine regulated the production of HSAF and analogs in a concentration-dependent manner. To verify the role of spermidine, we deleted LeSDC and LeADC genes, encoding S-adenosylmethionine decarboxylase and arginine decarboxylase, respectively, that are the key enzymes for spermidine biosynthesis. Both deletion mutants produced barely detectable spermidine and HSAF including its analogs, whereas the antifungals production was restored by exogenous spermidine. The results showed that the OH11 cells must maintain a proper spermidine homeostasis for the antifungals production. Indeed, the expression level of the key HSAF biosynthetic genes was significantly impaired in LeSDC and LeADC mutants, and exogenous spermidine restored the gene expression level in the mutants. Ornithine is a key substrate for HSAF biosynthesis, and OH11 genome contains arg1 and arg2 genes, encoding arginases that convert arginine to ornithine. While the expression of arg1 and arg2 was affected slightly upon mutation of LeSDC and LeADC, exogenous spermidine significantly increased the arginase gene expression in LeSDC and LeADC mutants. Together, the data revealed a previously unrecognized mechanism, in which spermidine controls antibiotic production through controlling both the biosynthetic genes and the substrate-production genes.

Impact of Trypanosoma cruzi infection on nitric oxide synthase and arginase expression and activity in young and elderly mice

Elderly organisms are more susceptible to infectious diseases. However, the impact of aging on antiparasitic mechanisms, especially the nitric oxide pathway, is poorly understood. Using an integrated in vivo and in vitro model, we compared the severity of Trypanosoma cruzi infection in young and elderly (8 or 72 weeks old) mice. Forty C57BL/6 mice were randomized into four groups: Y-inf, young infected; Yn-inf, young uninfected; A-inf, aged infected; An-inf, aged uninfected. Parasitemia was measured daily, and animals were euthanized after 15 days of infection. Trypanosoma cruzi-induced inflammatory processes were analyzed in blood and heart samples, as well as in bone marrow-derived macrophages (BMDMs) co-cultured with splenocytes isolated from young or elderly mice. Our results indicated upregulated IgG2b and IL-17 production in elderly animals, which was not sufficient to reduce parasitemia, parasitic load and myocarditis to levels observed in young animals. The higher susceptibility of elderly mice to T. cruzi infection was accompanied by reduced cardiac inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) and IFN-γ levels, as well as an antagonistic upregulation of arginase-1 expression and arginase activity. The same responses were observed when BMDMs co-cultured with splenocytes from elderly mice were stimulated with T. cruzi antigens. Our findings indicate that elderly mice were more susceptible to T. cruzi infection, which was potentially related to an attenuated response to antigenic stimulation, inhibition of iNOS gene expression and NO production, and antagonistic upregulation of arginase gene expression and activity, which created favorable conditions for heart parasitism and myocarditis development.

The Expression Pattern of &lt;i&gt;ArgAH1&lt;/i&gt; and &lt;i&gt;ArgAH2&lt;/i&gt; Genes in &lt;i&gt;Arabidopsis thaliana&lt;/i&gt;

Urea is the major nitrogen form supplied as fertilizer in agricultural production and also an important nitrogen metabolite in plants. Urea can be generated from Arginine breakdown by Arginase in germinating seeds under salt stress. To better understanding the biological functions of Arginase gene, here, we carried out the expression analysis of ArgAH1 and ArgAH2 genes in response to salt stress and different nitrogen source during early seed germination and seedling growth stage. Our results show that ArgAH1 and ArgAH2 were highly expressed in root and siliques of Arabidopsis under normal conditions, respectively. However, the expression levels of ArgAH1 and ArgAH2 was up-regulated by salt stress in both roots and cotyledons. In addition, the expression levels of ArgAH1 and ArgAH2 were also found to response to different nitrogen sources (e.g. NH 4 Cl, urea, arginine), different expression pattern are shown in both the roots and cotyledons under these treatment conditions. These results suggest that two arginase genes have different biological function in response to abiotic stresses in plants.

Association of increased risk of asthma with elevated arginase & interleukin-13 levels in serum & rs2781666 G/T genotype of arginase I.

Background & objectives:High expression of arginase gene and its elevated level in serum and bronchial lavage reported in animal models indicated an association with the pathogenesis of asthma. This study was undertaken to assess the serum arginase activity in symptomatic asthma patients and healthy controls and to correlate it with cytokine levels [interleukin (IL)-4 and IL-13] and arginase I (ARG1) gene polymorphism.Methods:Asthma was confirmed by lung function test according to the GINA guidelines in patients attending Allergy and Pulmonology Clinic, Bhagwan Mahavir Hospital and Research Centre, Hyderabad, India, a tertiary care centre, during 2013-2015. Serum arginase was analyzed using a biochemical assay, total IgE and cytokine levels by enzyme-linked immunosorbent assay and genotyping of ARG1 for single-nucleotide polymorphisms (SNPs) rs2781666 and rs60389358 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.Results:There was a significant two-fold elevation in the arginase activity in asthmatics as compared to healthy controls which correlated with disease severity. Non-atopic asthmatics showed elevated activity of arginase compared to atopics, indicating its possible role in intrinsic asthma. Levels of serum IL-13 and IL-4 were significantly high in asthma group which correlated with disease severity that was assessed by spirometry. A positive correlation was observed between arginase activity and IL-13 concentration. Genetic analysis of ARG1 SNPs revealed that rs2781666 G/T genotype, T allele and C-T haplotype (rs60389358 and rs2781666) were associated with susceptibility to asthma.Interpretation & conclusions:This study indicated that high arginase activity and IL-13 concentration in the serum and ARG1 rs2781666 G/T genotype might increase the risk of asthma in susceptible population. Further studies need to be done with a large sample to confirm these findings.

Comprehensive molecular analysis of arginase-encoding genes in common wheat and its progenitor species

Arginase (ARG) contributes to nitrogen remobilization by conversion of arginine to ornithine and urea. However, wheat ARG genes have not yet been identified. Here we isolated and characterized ARG genes from wheat and its progenitor species and found that a single copy was present in wheat progenitors. Three common wheat ARG genes of TaARG-2AS, TaARG-2BS, and TaARG-2DS were experimentally assigned to the short arms of the group 2 chromosomes. We found an in-frame stop codon in TaARG-2AS, but not in the other two genes. The highest expression was detected in stems and sheaths for TaARG-2BS and in leaves for TaARG-2DS. Both genes have similar expression trend in different developmental stages, peaking at booting and grain filling stages. TaARG-2BS transcript was induced by high salinity and drought, whereas TaARG-2DS was induced by drought only, but neither of them were induced by low temperature. In addition, both genes showed analogous expression pattern upon powdery mildew (PM) infection in the resistant line Pm97033, with TaARG-2BS induced greatly at 72 h post PM infection. In contrast, no obvious transcripts were accumulated for TaARG-2DS in the PM susceptible line Wan7107. Monocot ARGs have more conserved mitochondrion-targeting signals and are more evolutionarily conserved than dicot ARGs.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome.

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg R.pyc cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg R.pyc cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

Isolation and characterization of monocyte/macrophage from peripheral blood of half smooth tongue sole (Cynoglossus semilaevis)

In the present study, the peripheral blood cells of half smooth tongue sole (Cynoglossus semilaevis) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function.

Characterisation of arginase paralogues in salmonids and their modulation by immune stimulation/ infection

In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.

Decreased Rate of Plasma Arginine Appearance in Murine Malaria May Explain Hypoargininemia in Children With Cerebral Malaria

Plasmodium infection depletes arginine, the substrate for nitric oxide synthesis, and impairs endothelium-dependent vasodilation. Increased conversion of arginine to ornithine by parasites or host arginase is a proposed mechanism of arginine depletion. We used high-performance liquid chromatography to measure plasma arginine, ornithine, and citrulline levels in Malawian children with cerebral malaria and in mice infected with Plasmodium berghei ANKA with or without the arginase gene. Heavy isotope-labeled tracers measured by quadrupole time-of-flight liquid chromatography-mass spectrometry were used to quantify the in vivo rate of appearance and interconversion of plasma arginine, ornithine, and citrulline in infected mice. Children with cerebral malaria and P. berghei-infected mice demonstrated depletion of plasma arginine, ornithine, and citrulline. Knock out of Plasmodium arginase did not alter arginine depletion in infected mice. Metabolic tracer analysis demonstrated that plasma arginase flux was unchanged by P. berghei infection. Instead, infected mice exhibited decreased rates of plasma arginine, ornithine, and citrulline appearance and decreased conversion of plasma citrulline to arginine. Notably, plasma arginine use by nitric oxide synthase was decreased in infected mice. Simultaneous arginine and ornithine depletion in malaria parasite-infected children cannot be fully explained by plasma arginase activity. Our mouse model studies suggest that plasma arginine depletion is driven primarily by a decreased rate of appearance.

Cloning and comparative analysis the proximal promoter activities of arginase and agmatinase genes in Apostichopus japonicus

Our previous work demonstrated that Apostichopus japonicus arginase and agmatinase from l-arginine metabolism synergistically compete with NOS under pathogens challenge. Here we conducted a study to further investigate the mechanism in the regulation of arginase and agmatinase genes in l-arginine metabolism using EPC cell system. Luciferase analysis and progressive 5' deletion analysis suggested that Ajagmatinase promoter was a very robust promoter for its transcription, and the core region of Ajarginase promoter was located within −277 bp to −157 bp. Besides, their promoter activities were significantly activated by LPS and l-arginine challenge both in a time- and dose-dependent manners in EPC cells. When different truncated reporter vector and expression vector co-transfection experiment revealed transcription factor NF-κB/Rel and STAT5 could significantly inhibited Ajarginase promoter activity, but not Ajagmatinase. Our findings were provided novel insights into the transcriptional regulation of Ajarginase and Ajagmatinase, and selectively change their expressions might prevent pathogens infection.

Involvement of Arginase in Methyl Jasmonate–induced Tomato Fruit Chilling Tolerance

The physiological role of arginase in nitrogen remobilization processes from protein degradation during seed germination has well been described in several species. However, very little is known about its possible roles in plant stress responses. Treatment of tomato fruit (Solanum lycopersicum L.) with 0.05 mm methyl jasmonate (MeJA) enhanced transcription levels of arginase genes, especially LeARG2. Chilling injury (CI) of fruit treated with 0.05 mm MeJA for 12 hours was also effectively alleviated, as manifested by decreases in CI index, electrolyte leakage, and malondialdehyde (MDA) content. To investigate the potential role of arginase in MeJA-induced chilling tolerance, fruit were treated with MeJA or the arginase inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) combined with MeJA and then stored at 2 °C for 28 days. MeJA-induced arginase activity was strongly inhibited and the reduction of CI by MeJA was nearly abolished by the inhibitor. In addition, MeJA treatment increased the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX); inhibited peroxidase (POD) activities; and promoted proline and polyamines accumulation. These effects were partially counteracted by nor-NOHA; putrescine accumulation, however, was unaffected by the inhibitor. Our results indicate that arginase may be involved in MeJA-induced chilling tolerance, possibly by ameliorating the antioxidant enzyme system of fruit and increasing proline levels.

Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings.

Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene OsARG is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed OsARG into upland cotton R18 by Agrobacterium-mediated genetic transformation and analyzed the function of OsARG in transgenic cotton. Two independent OsARG expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the OsARG gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the OsARG gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG-26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the OsARG gene could potentially be used to improve cotton fiber length traits.

Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when 13C6-arginine (Arg-6) is used for labeling, it is less successful when 13C6 15N4-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of 13C5 15N2-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.

Integration of nondegradable polystyrene and degradable gelatin in a core-sheath nanofibrous patch for pelvic reconstruction.

Pelvic organ prolapse (POP) is a serious health issue affecting many adult women. Complications of POP include pelvic pressure, pelvic pain, and problems in emptying their bowels or bladder. Sometimes, POP may even cause urinary outflow obstruction and lead to bladder or kidney infections. Currently, synthetic and naturally derived materials have been chosen for treatment of POP to reduce the high recurrence rates after surgical interventions. However, existing materials for POP treatment cannot meet the clinical requirements in terms of biocompatibility, mechanics, and minimal risk of rejection. Especially, erosion in synthetic polymers and rapid degradation in natural polymers limit their further applications in clinics. To address these concerns, we report a novel POP replacement using core–sheath polystyrene/gelatin electrospun nanofiber mesh. The outside gelatin sheath provides a hydrophilic surface and implantable integrity between host and guest, while the inner PS core offers the necessary mechanical support. The composite mesh shows graft accommodation in pelvic submucosa after implantation in vivo, as shown in hematoxylin–eosin staining and T helper cell phenotype and macrophage phenotype stainings. Qualitative analysis of inducible nitric oxide synthase, arginase, interferon-γ, and interleukin-10 gene expressions also indicates that the implanted composite mesh switches to accommodation mode 2 weeks postimplantation. Thus, these novel core–sheath polystyrene/gelatin nanofibrous membranes are promising in pelvic reconstruction.

An alternative, arginase‐independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.

Expression Analysis of &lt;i&gt;Arabidopsis thaliana&lt;/i&gt; Genes &lt;i&gt;ARGAH1&lt;/i&gt;、&lt;i&gt;ARGAH2&lt;/i&gt; and the Response to Sodium Chloride Stress During Seed Germination

Arabidopsis thaliana possesses two arginase-encoding genes（ ARGAH1 、 ARGAH2 ）, which catalyses the catabolism of arginine into L-ornithine and urea. In this research, we focus on the function of the two genes ( ARGAH1 、 ARGAH2 ) encoding Arabidopsis thaliana arginase. The cDNA of cloned ARGAH1 is 1347 bp, and 5' untranslated region (UTR) is 145 bp and the 3' UTR is 166 bp. The open reading frame of ARGAH1 is 1029 bp, encoding 342 amino acids. ARGAH2 is 1284 bp, and 5' untranslated region (UTR) is 34 bp and the 3' UTR is 215 bp. The open reading frame of ARGAH2 is 1035 bp, encoding 344 amino acids. We studied the different expression models of arginase genes under NaCl stress by qRT-PCR and other biotechnology. Interestly, we observed that the expression of ARGAH1 and ARGAH2 is both induced by NaCl stress. At the same time, the enzyme assay of argianse also showed an increase during seed germination. Therefore, we maintain that the argianse genes play a role against to NaCl stress especially during the germination.

Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats

Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77days for 25days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p<0.05) food intake, but retarded (p<0.05) growth, increased (p<0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p<0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p<0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p>0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism.

Arginase as a mediator of diabetic retinopathy

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.