Hydrolysis Of Arginine Research Articles

Screening and characterization of exopolysaccharide producing Lactobacillus spp.

The present study was undertaken with the objective to screen promising L. gasseri, L. reuteri and L. helveticus isolates for the Exopolysaccharide (EPS) production and their characterization through biochemical and molecular tests. A total of 7 isolates of L. gasseri and 37 isolates of L. reuteri and one isolate of L. helveticus were screened for the EPS production through various tests and finally L . gasseri Lg62 and L. helveticus V3 were selected for the further study. The most promising EPSs producing isolates i.e. V3 and Lg62 gave a prominent growth on RR-MRS agar plate whereas Lg63 and Lg50A were not so prominent on RR-MRS agar plate. V3 and Lg62 showed better viscosity than Lg63 and Lg50A. V3 showed viscosity and corresponding O.D. in the range of 1.100 -1.677 cp and 0.753-1.207 (at 525nm) respectively at 42 0 C and 1.050-1.242 cp and 0.673-0.952 (at 525nm) at 37 0 C respectively. The isolates were found to be purple coloured Gram-positive rods of varying size under microscope. Lactobacillus cultures were catalase negative. V3 showed growth when incubated in MRS broth at 37 0 C as well as at 42 0 C for 24-48 hrs, in terms of turbidity whereas there was no turbidity at 15 0 C. In the arginine hydrolysis test neither LG62 nor V3 showed positive reaction. Both the EPS+ cultures produced acid from glucose, maltose, fructose and sucrose on incubation at 37 0 C for 24 hours. None of the culture produced gas in glucose. Both the cultures V3 and Lg62 showed growth at 4% NaCl concentration and changed the colour of the media from purple to yellow. Growth of V3 was not detected in 6.5% NaCl concentration whereas Lg62 showed growth in the same concentration. After identification of the selected cultures by biochemical techniques they were further confirmed by PCR using species specific primers.

AmcA-a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis.

Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis (SB) depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine. In this study, we demonstrate a role of the putative mitochondrial transporter AmcA (AFUA_8G02760) in SB of A. fumigatus. Consistent with a role in cellular ornithine handling, AmcA-deficiency resulted in decreased cellular ornithine and arginine contents as well as decreased siderophore production on medium containing glutamine as the sole nitrogen source. In support, arginine and ornithine as nitrogen sources did not impact SB due to cytosolic ornithine availability. As revealed by Northern blot analysis, transcript levels of siderophore biosynthetic genes were unresponsive to the cellular ornithine level. In contrast to siderophore production, AmcA deficiency did only mildly decrease the cellular polyamine content, demonstrating cellular prioritization of ornithine use. Nevertheless, AmcA-deficiency increased the susceptibility of A. fumigatus to the polyamine biosynthesis inhibitor eflornithine, most likely due to the decreased ornithine pool. AmcA-deficiency decreased the growth rate particularly on ornithine as the sole nitrogen source during iron starvation and sufficiency, indicating an additional role in the metabolism and fitness of A. fumigatus, possibly in mitochondrial ornithine import. In the Galleria mellonella infection model, AmcA-deficiency did not affect virulence of A. fumigatus, most likely due to the residual siderophore production and arginine availability in this host niche.

Identification and Characterization of Lactic Acid Bacteria Involved in Natural and Lactic Acid Bacterial Fermentations of Pastes of Soybeans and Soybean-Maize Blends Using Culture-Dependent Techniques and Denaturing Gradient Gel Electrophoresis

Pastes of soybeans and soybean-maize blends were fermented with inoculum through back-slopping using lactic acid bacteria (LAB) fermented cereal gruel, thobwa and without inoculum (naturally). LAB involved in the fermentations were characterized using culture-dependent and culture-independent analyses. Decreases in pH from 6.4 to 3.9–4.2 and from 6.9 to 5.4–5.8 after 72 h were observed in LAB fermented pastes (LFP) and in naturally fermented pastes (NFP), respectively. LAB increased from 5.0 to 8.7–9.6 log10 cfu/g in NFP and from 8.1 to 9.3 log10 cfu/g in LFP. LAB in both fermentations were heterofermentative lactobacilli (82.4%) and homofermentative cocci (17.6%), of which 44.7% and 42.9% were exopolysaccharide producers, respectively. Principal component analysis based on carbohydrate fermentation, CO2 production and arginine hydrolysis showed four clusters dominated by Lactobacillus fermentum, Weissella confusa, Lactobacillus brevis 1 and Pediococcus pentosaceus, respectively. Sequencing of 16S rDNA gene confirmed Lb. fermentum, W. confusa/W. cibaria, and P. pentosaceus as identities of species in three clusters. Denaturing gradient gel electrophoresis (DGGE) confirmed these species as the dominant microbiota. DGGE showed higher similarity in microbial profiles of LFP throughout fermentation and low similarity in NFP during early and late stages of fermentation.

Selection of autochthonous strains as starter cultures for fermented fish products.

This paper was the 1st research focusing on the design of a halophilic lactic starter for the production of fermented fish products using a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising strains. Fifty-nine strains were randomly selected from salted fish and phenotypically characterized through Gram staining, catalase activity, glucose metabolism, H2 S and indole production, nitrate reduction, citrate utilization, and hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea. Then the Gram positive isolates (44 out of 59) were studied for their growth at different temperatures (10, 25, 40, and 55 °C), salt (0%, 20%, and 30%), pHs (4.5 and 9.5), and acidification score in lab medium. Data were modelled through growth index and used as input to run a preliminary cluster analysis and a principal component analysis. Three promising strains were selected, identified as members of the genus Pediococcus and used for the validation at laboratory level through the assessment of their performances in the production of a fermented fish sauce. The results were really promising as their use not only reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. This paper represents a 1st report on the use of a simple step-by-step methodology to select promising halophilic strains for the optimization of a starter for fish-fermented products.

Arginase-1 is a novel immunohistochemical marker of hepatocellular differentiation.

Arginase-1 is an enzyme that catalyzes the hydrolysis of arginine to ornithine and urea in the urea cycle. In normal tissues, arginase-1 is primarily expressed in hepatocytes. Recent investigations have reported that the vast majority of hepatocellular carcinomas express this marker, but it is found only rarely in nonhepatocellular tumors. Owing to its restricted expression in hepatocellular carcinomas, arginase-1 has proved to be a useful immunohistochemical marker for assisting in distinguishing between these tumors and other neoplasms with which they may be confused.

Crystallization of dicalcium phosphate dihydrate with presence of glutamic acid and arginine at 37 °C.

The formations of non-metabolic stones, bones and teeth were seriously related to the morphology, size and surface reactivity of dicalcium phosphate dihydrate (DCPD). Herein, a facile biomimetic mineralization method with presence of glutamic acid and arginine was employed to fabricate DCPD with well-defined morphology and adjustable crystallite size. In reaction solution containing more arginine, crystallization of DCPD occurred with faster rate of nucleation and higher density of stacked layers due to the generation of more OH(-) ions after hydrolysis of arginine at 37 °C. With addition of fluorescein or acetone, the consumption of OH(-) ions or desolvation reaction of Ca(2+) ions was modulated, which resulted in the fabrication of DCPD with adjustable crystallite sizes and densities of stacked layers. In comparison with fluorescein-loading DCPD, dicalcium phosphate anhydrate was prepared with enhanced photoluminescence properties due to the reduction of self-quenching effect and regular arrangement of encapsulated fluorescein molecules. With addition of more acetone, DCPD was prepared with smaller crystallite size via antisolvent crystallization. The simulated process with addition of amino acids under 37 °C would shed light on the dynamic process of biomineralization for calcium phosphate compounds.

English

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. The role of arginase in insect has been seen in the growth of insect at various stages of development and energy requirement for flight. Here, we reported the properties of arginase in the gut of adult cockroach, Periplaneta americana. The enzyme was partially purified with ammonium sulphate precipitation and affinity chromatography. The specific activity of the arginase was 3.0 µmol/min/mg of protein. The Michealis (Km) constant was 0.33 mM and the arginase preferred arginine as substrate. The optimum pH was 7.0, while the optimum temperature was 80°C. The ascorbic acid, reduced glutathione (GSH) and 2-mercaptoethanol completely inactivated the enzyme. The amino acids: lysine, valine, serine, and asparagine showed moderate inhibition (with residual arginase activities of 81.7, 96.1, 96.9 and 97.0%, respectively), while proline and cysteine (>100%, respectively) stimulated the arginase activity. The cations: Sn2+, Hg2+, Ni2+ and Co2+ showed slight inhibition on the enzyme. The enzyme was markedly enhanced by Zn2+ and Mg2+. Key words: Arginase, insect gut, cockcroach, characterization.&nbsp

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis

IntroductionIn some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. MethodsMDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. ResultsCML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). ConclusionsMDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures:No relevant conflicts of interest to declare.

Abstract C32: Metabolic advantages of urea cycle misregulation in renal cell carcinoma

Abstract Background: The urea cycle is the central pathway involved in the detoxification of free ammonia species in mammals, which takes place at two sites: the liver and the kidney. Renal cell carcinoma is the most common type of kidney malignancy and the main subtype, clear cell renal cell carcinoma (ccRCC), makes up 75% of these cancers. ccRCC is distinguished by both radio- and chemo-resistance and treatment options for this disease are limited. Argininosuccinate synthase 1 (ASS1) and arginase II (ARG2) are two key enzymes in the urea cycle, the former being responsible for the synthesis of arginine while the latter hydrolyzes arginine to ornithine in the last step of the cycle. We have shown that in vitro, both normal kidney and ccRCC cell lines require exogenous arginine for growth and this suggests that increased arginine availability contributes to the pathogenesis of ccRCC. In this study, we measured expression changes of ASS1 and ARG2, as well as other enzymes involved in arginine metabolism, in ccRCC versus normal kidney tissue from patient samples to determine the metabolic advantages of an altered urea cycle. Our findings highlight the applicability of targeting cancer metabolism of a specific nutrient to reduce the proliferative capacity of ccRCC tumors. Methods: This study utilized both normal kidney and ccRCC cell lines to analyze the expression levels of four enzymes involved in arginine metabolism and the urea cycle. These results were confirmed in matched normal and ccRCC primary patient samples obtained from the Cooperative Human Tissue Network (CHTN, n=26) using both qPCR analysis for mRNA and immunohistochemistry for the levels of protein in these tissues. Moreover, our results were validated on a large-scale analysis of gene expression in ccRCC tumors via bioinformatic analysis of data available from The Cancer Genome Atlas (TCGA, n=480). We are currently manipulating the levels of ARG2 and ASS1 in normal kidney and ccRCC cell lines to elucidate metabolic advantages in proliferation and colony formation. Furthermore, we are working toward metabolomic analysis of primary patient samples of ccRCC tissue to discover additional altered metabolites of the urea cycle in this setting. Results: The expression levels of ASS1 and ARG2 are decreased 4-fold and 7-fold, respectively, based on analysis of ccRCC TCGA data. Additionally, analysis of matched tissue obtained from the CHTN mirrors this data, with the majority of ccRCC samples (&amp;gt;85%) under-expressing at least one of these enzymes. Moreover, the arginine metabolizing enzyme glycine amidinotransferase is 3-fold under-expressed and the arginine transporter CAT-1 is 10-fold over-expressed in ccRCC. Cumulatively, these data point to an altered metabolism of arginine in ccRCC, with the modified enzyme levels leading to an increased level of arginine to support growth and biosynthesis of ccRCC tumors. Conclusion: Our data demonstrate that arginine metabolism is altered in ccRCC tumors versus normal kidney tissue and that this increased arginine availability likely enables sustained proliferation of the tumor tissue. This study highlights the applicability of targeting cancer metabolism of a specific amino acid, arginine, as a possible therapeutic intervention for ccRCC. Citation Format: Joshua Ochocki, Nan Lin, Bo Qiu, M. Celeste Simon. Metabolic advantages of urea cycle misregulation in renal cell carcinoma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C32.

Isolation and kinetic properties of arginase in the gut of grasshopper (&lt;i&gt;Zonocerus variegatus&lt;/i&gt; Linn)

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Arginase was purified and characterized from the gut of Zonocerus variegatus through DEAE-cellulose and biogel-P100 gel filtration chromatography. The specific activity of the enzyme was 3.7 μmoles/min per mg of protein and a yield of 14.7%. An apparent molecular weight of 143,000 daltons was estimated by gel filtration on biogel P-100. The Michaelis constant (Km) of the enzyme was 40 mM with arginine as substrate. The optimum pH was 8.0 and the optimum temperature was 40 oC for Z. variegatus arginase. The enzyme was stable up to 40 oC for 20 min and lost all of its activity at 80 oC. The enzyme was specific for arginine as substrate. The enzyme was strongly enhanced in the presence of Mn2+, Na+, NH4 + and Hg2+ showed similar activation. Ni2+ and Zn2+ slightly inhibited Z. variegatus. Chelating (EDTA, citrate, ascorbic acid and urea) and thio (2-mercaptoethanol and cystein) compounds inhibited the activity of arginase in Z. variegatus. While amino acids (proline, lysine, aspartate and valine) showed no inhibition on arginase activity. The presence of arginase in the gut of Zonocerus variegatus could be for other functions rather than urea production in urea cycle. © 2013 International Formulae Group. All rights reserved. Keywords: Arginase; Zonocerus variegatus; Distribution; Physicochemical Properties.

Characterization of the Link between Ornithine, Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.

Kinetics of the utilization of dietary arginine for nitric oxide and urea synthesis: insight into the arginine–nitric oxide metabolic system in humans

The systemic availability of oral/dietary arginine and its utilization for nitric oxide (NO) synthesis remains unknown and may be related to a competitive hydrolysis of arginine into urea in the splanchnic area and systemic circulation. We investigated the kinetics and dose-dependency of dietary arginine utilization for NO compared with urea synthesis and studied the characteristics of the arginine-NO metabolic system in healthy humans. We traced the metabolic fate and analyzed the utilization dynamics of dietary arginine after its ingestion at 2 nutritional amounts in healthy humans (n = 9) in a crossover design by using [(15)N-(15)N-(guanido)]-arginine, isotope ratio mass spectrometry techniques, and data analysis with a compartmental modeling approach. Whatever the amount of dietary arginine, 60 ± 3% (±SEM) was converted to urea, with kinetics indicative of a first-pass splanchnic phenomenon. Despite this dramatic extraction, intact dietary arginine made a major contribution to the postprandial increase in plasma arginine. However, the model identified that the plasma compartment was a very minor (~2%) precursor for the conversion of dietary arginine into NO, which, in any case, was small (<0.1% of the dose). The whole-body and plasma kinetics of arginine metabolism were consistent with the suggested competitive metabolism by the arginase and NO synthase pathways. The conversion of oral/dietary arginine into NO is not limited by the systemic availability of arginine but by a tight metabolic compartmentation at the systemic level. We propose an organization of the arginine metabolic system that explains the daily maintenance of NO homeostasis in healthy humans.

OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice.

Nitrogen is a crucial nutrient for plant growth and development. Arginine is considered to be an important amino acid for nitrogen transport and storage, playing a crucial role during plant seedling development. However, little is known about the role of arginine in nitrogen remobilization at the reproductive stage. We isolated a rice mutant nglf-1 with reduced plant height, small panicle and grain size, and low seed-setting rate (10% in nglf-1 compared to 93% in wild-type). Map-based cloning revealed that the mutant phenotype was caused by loss of function of a gene (OsARG) encoding an arginine hydrolysis enzyme, which is consistent with arginine accumulation in the mutant. The phenotype was partially corrected supplying exogenous nitrogen, and fully corrected by expression of a wild-type OsARG transgene. Over-expression of OsARG in rice (cv. Kitaake) increased grain number per plant under nitrogen-limited conditions. OsARG was ubiquitously expressed in various organs, but most strongly in developing panicles. The OsARG protein was localized in the mitochondria, consistent with other arginases. Our results suggest that the arginase encoded by OsARG, a key enzyme in Arg catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous nitrogen. OsARG is a potential target for crop improvement.

Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity.

AbstractAbstract 2779 Background:Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. Aim:The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients. Methods:MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results:CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum. Conclusions:Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion. Disclosures:Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.

Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.

Characterization and Selection of LAB Isolated from Inner Mongolia Traditional Meat Sausages

A total of 69 gram-positive, catalase-negative strains isolated from Inner Mongolia traditional meat sausages were characterized in respect to their technological properties, including acid production, salt and nitrite tolerance, gas, slime and H2S production, hydrolysis of arginine and biogenic amine production. All of the isolated strains decrease the pH below 5.0 after 24h of cultivation. All isolates are able to grow with 6.5% NaCl or with 150mg L-1NaNO2and 51 isolates are able to grow with 10% NaCl. All isolates don’t produce gas and slime. 54 isolates don’t produce H2S. 23 isolated strains don’t produce NH3 from arginine, and 40 isolates don’t produce biogenic amine. Consolidation of these results, 8 strains show the best technological properties to be used as starter culture for sausage production. The bacterial selected are then subjected to biochemical tests using the API 50 CH and they are identified as 6 Lactobacillus planarum and 2 Lactobacillus curvatus.

Immunohistochemical study of arginase-1 in the spinal cords of rats with clip compression injury

The expression of arginases, enzymes that catalyze the hydrolysis of arginine to ornithine and urea, was studied in the inflammatory lesions of spinal cord injury (SCI) in rats. The level of arginase-1 expression in rat spinal cords with clip compression injury was determined by Western blot analysis and immunohistochemistry. Western blot showed that the level of arginase-1 increased in the core lesion of SCI at day 1 post injury and continued to increase through days 4 (p<0.05) and 7 (p<0.01). Immunohistochemical analysis showed that arginase-1 was constitutively expressed in neurons and glial cells in sham control spinal cords. In SCI lesions, arginase-1 was additionally detected in inflammatory cells, particularly in isolectin B4-positive macrophages and reactive astrocytes within the core lesion. These findings suggest that the increased level of arginase-1 in SCI is associated with an increase in macrophages and reactive astrocytes, possibly contributing to the modulation of inflammation during the course of SCI.

Screening and Characterisation ofPseudomonas fluorescence: A Biological Control Agent against Wilt Fungus inCajanus cajan(L.) Millsp.

About 310 Pigeon pea (Cajanus cajan (L.) Millsp.) rhizobacteria were isolated from cultivated soils of North-East India (Alwar district, Rajasthan). Among the total isolates, 25 isolates were confirmed as Pseudomonas fluorescence based on the biochemical tests, such as arginine hydrolysis, catalase activity, production of fluorescing compounds, gelatin liquefaction and growth at 4°C and 42°C. Their antifungal activity against wilt fungus Fusarium udum (strain of pigeon pea) was examined in vitro and in vivo. Survivality of the isolates under stress conditions of temperature and pH was examined. Five strains (BISR-E, BISR-T, BISR-V, BISR-X and BISR-Y) gave maximum temperature tolerance and four strains (BISR-B, BISR-C, BISR-D and BISR-H) could survive under variable pH range 4–10. Twelve isolates of P. fluorescence were exhibited strong antifungal activity against F. udum. Zone of inhibition was measured. Twelve isolates were observed with more than 30% RI value with maximum RI value recorded in strain BISR-W and BISR-N (42.0%). Net house pot studies screening results revealed that 17 isolates (BISR-A, BISR-B, BISR-C, BISR-D, BISR-E, BISR-G, BISR-H, BISR-I, BISR-K, BISR-L, BISR-N, BISR-O, BISR-P, BISR-R, BISR-U, BISR-W and BISR-X) were observed as good plant growth-promoting rhizobacteria and biological control agents. Among these strains, isolate numbers BISR—B, BISR-F, BISR-K, BISR-N, BISR-O, BISR-P, BISR-W and BISR-X were proved themselves superior isolates having good biocontrol potential to manage F. udum in Pigeon pea plant and to enhance plant growth parameters to a significant level. Out of 25 isolates, 20 isolates (BISR-A, BISR-B, BISR-C, BISR-D, BISR-F, BISR-G, BISR-H, BISR-I, BISR-J, BISR-K, BISR-L, BISR-M, BISR-N, BISR-O, BISR-P, BISR-Q, BISR-R, BISR-S, BISR-T and BISR-V) could produce siderophore.

Immunohistochemical study of arginase 1 and 2 in various tissues of rats

Arginase 1 and arginase 2 catalyze the hydrolysis of arginine to ornithine and urea. The localization of these enzymes was studied in various tissues in Sprague–Dawley rats by immunohistochemistry and Western blotting. Western blot analysis showed that both arginase 1 and 2 were differentially expressed in the various organs examined. Arginase 1 was expressed at high levels in the liver, at moderate levels in the pancreas, and at low levels in the cerebrum, cerebellum, spinal cord, stomach, small and large intestines, kidneys, lungs, and spleen. The levels of arginase 2 immunoreactivity were high in the kidneys and pancreas, and moderate in the cerebrum, spinal cord, stomach, small intestine, large intestine, and lungs; the levels were very low in the liver and spleen compared with that in the cerebellum. Immunohistochemical analysis largely confirmed the results of the Western blot analysis. These findings indicate that the levels of arginase 1 and 2 varied among organs, suggesting that the arginase isoforms may play organ-specific roles in the urea cycle.

The Effect of Montelukast Sodium on Serum Arginase Levels in Patients with Seasonal Allergic Rhinitis

Nitric oxide (NO) imbalance appears to be important in the pathogenesis of allergic rhinitis. NO is synthesized from l-arginine by NO synthase (NOS). Competing with NOS for l-arginine is arginase, which catalyzes the hydrolysis of arginine to urea and ornithine. Therefore, increased serum arginase activity could potentially limit NO production catalyzed by inducible NOS, thus contributing to allergic rhinitis. This study was designed to investigate the effect of the cysteinyl leukotriene type 1 receptor antagonist, montelukast sodium on serum arginase levels in patients with seasonal allergic rhinitis. Twenty-five patients with seasonal allergic rhinitis (SAR; treatment group) and 16 nonasthmatic patients without allergic rhinitis (control group) were included in the study. Serum arginase levels and the mean total nasal symptoms scores were measured before and after oral montelukast sodium (10 mg) was administered daily for 4 weeks to the treatment group. Serum arginase levels and the mean total nasal symptoms scores were significantly lower in the treatment group after montelukast sodium administration compared with the baseline levels (p = 0.001). Serum arginase levels were significantly lower in the treatment group compared with the control group (p = 0.01). There was no statistically significant difference between the serum arginase levels of the treatment group before treatment and the control group (p = 0.05). There was a weak correlation between the mean total nasal symptoms scores and serum arginase levels in the treatment group before montelukast sodium administration (rs = 0.40; p = 0.05). Montelukast sodium may reduce serum arginase levels and total nasal symptoms scores of patients with SAR. Additional studies that compare the effectiveness of nasal corticosteroid and montelukast sodium on serum arginase levels should be conducted.