Argininosuccinate Lyase Research Articles

Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G 2/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

Population screening in a Druze community: the challenge and the reward

PurposeThe Druze community is characterized by consanguinity and endogamy, and by reluctance to genetic testing and technological interventions for the prevention of birth defects. Multiple patients with four rare and severe inborn errors of metabolism cerebrotendinous xanthomatosis, prolidase deficiency, argininosuccinate lyase deficiency, and carbamyl phosphate synthetase I deficiency were identified in an isolated Druze village in northern Israel. The aims of this study were to identify couples at risk for four inherited diseases, and to prevent birth defects in a community presenting religious and cultural obstacles to genetic testing. MethodsA genetic screening and counseling program in a high-risk community. ResultsThe 1425 residents who attended group genetic counseling sessions between 2003 and 2007 consented to genetic testing. We identified 217 carriers for either one or two disease causing mutations. High carrier frequencies for cerebrotendinous xanthomatosis, prolidase deficiency, argininosuccinate lyase deficiency, and carbamyl phosphate synthetase I deficiency were identified as 1:11, 1:21, 1:41, and 1:95, respectively. Fifty-eight percent (125) of the carriers' spouses agreed to genetic counseling and testing. Ten couples at risk for affected offspring were identified and offered prenatal genetic counseling and diagnosis. ConclusionsThe genetic screening program, the first of its kind reported in a Druze community, was well received. We expect this program to increase awareness of genetic counseling, to contribute to disease prevention, and to serve as a model for other isolated communities.

Hereditary urea cycle diseases in Finland

To estimate the incidence of urea cycle diseases (UCDs) in Finland and determine the course of the various disorders as well as the outcome. The original data were collected in the years 1998-2001. The diagnoses made after 2001, as well as the current status of the patients, were updated by surveys in the spring of 2007. We found a total of 55 cases of UCDs in Finland by 2007: 30 cases of ornithine transcarbamylase (OTC) deficiency, 20 of argininosuccinate lyase (ASL) deficiency, 3 of carbamyl phosphate synthetase (CPS-I) deficiency, 1 of type 1 citrullinaemia and 1 of argininaemia. The estimated total incidence of UCDs was 1:39 000. The incidences of individual disorders were: OTC deficiency 1:62 000, ASL deficiency 1:144 000, CPS deficiency 1:539 000 and citrullinaemia 1:1 616 000. Eighteen (33%) of the patients with a diagnosis of UCD have died, most during their first hyperammonaemic crisis. One patient with OTC deficiency has had a liver transplant. Neurological symptoms of varying severity are common among these patients, particularly those with ASL deficiency. The first survey on the incidence of UCDs in Finland shows some differences in the occurrence rates compared to other countries. Hyperammonaemia, and the neurological symptoms caused by it, can be avoided in most patients with late-onset UCDs with a standard treatment. However, in patients with ASL deficiency, the development of neurological symptoms seems to be inevitable in spite of careful treatment and avoidance of hyperammonaemia.

Lactobacillus plantarum response to inorganic carbon concentrations: PyrR2-dependent and -independent transcription regulation of genes involved in arginine and nucleotide metabolism

Lactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO(2)-enriched air. At low inorganic carbon (C(i)) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to C(i) via a PyrR(2)-mediated mechanism. A previous study of high-CO(2)-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and C(i)-dependent nutritional needs. To better understand L. plantarum's adaptation to C(i) availability, additional C(i)-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to C(i) availability, suggesting that the C(i) response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in C(i) levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to C(i) availability, as previously demonstrated for the pyr genes. However, PyrR(2) was not essential for the C(i)-regulated transcription of argH and guaB, demonstrating that PyrR(2) modulates only a subset of C(i)-regulated genes. These results suggest that the C(i) response may involve at least two regulatory mechanisms in L. plantarum.

Genetic variation in the urea cycle: a model resource for investigating key candidate genes for common diseases

The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases.

Hyperammonaemia due to primary hyperparathyroidism-related renal tubular acidosis with incidental hypovitaminosis-D

Ammonia is produced by amino acid and protein catabolism and is toxic to brain and muscles. It is converted to urea in the liver by urea-cycle enzymes, which is then excreted through the kidneys. Hyperammonaemia may result from a genetic defect or deficiency of the urea-cycle enzymes, or acquired conditions such as Reye's syndrome, liver failure, high-dose chemotherapy and severe infection [1]. In the urea cycle, which takes place exclusively in periportal hepatocytes, six enzymes are involved in the conversion of ammonia to urea. These areN-acetylglutamate synthetase, carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase. A defect or deficiency of any of these enzymeswill result in hyperammonaemia [2]. Hyperammonaemia is most severe when the enzyme defect is in the early steps of the urea cycle such as carbamyl phosphate synthetase deficiency and ornithine transcarbamylase. Infection, trauma or ingestion of large amounts of proteins is usually the precipitating factor in causing hyperammonaemia. The clinical features of hyperammonaemia are usually nonspecific in adults and include vomiting, lethargy, sleep and behavioural disturbances, hallucinations, delusions and psychosis. Electroencephalogram may show an encephalopathic slow wave pattern [3]. Renal tubular acidosis (RTA) are a group of disorders of renal tubular function that lead to hyperchloremic metabolic acidosis in the presence of well-preserved glomerular function.

Polyaspartoyl· l-arginine inhibits platelet aggregation through stimulation of NO release from endothelial cells

Polyaspartoyl· l-arginine (PDR) is an inhibitor of platelet aggregation ex vivo but in vitro. This study attempts to elucidate the target cell of PDR action and its action mechanism. PDR (1.7–170 µg/ml) significantly inhibited platelet aggregation in vitro in the presence of rat aortic endothelial cells (RAEC), NO synthase inhibitor N-nitro- l-arginine methyl ester ( l-NAME) inhibited this effect, but it was ineffective in the RAEC absence. Correspondingly, PDR increased NO level in the supernatants of the platelet reactants in RAEC presence, but failed to influence NO level in RAEC absence, and these effects of PDR were more potent than those of l-arginine. Furthermore, PDR markedly elevated the intracellular level of l-arginine, and it (17–170 µg/ml) also augmented l-citrulline level in RAEC, argininosuccinate lyase (ASL) inhibitor succinate enhanced its effect on l-citrulline but l-NAME weakened it. 170 µg/ml of PDR slightly increased the l-aspartate level in RAEC, and succinate enhanced this effect. However l-arginine, l-aspartate or the combination of l-arginine and l-aspartate failed to change levels of these amino acids. In addition, PDR (170 μg/ml) stimulated the expression of argininosuccinate synthetase (ASS) protein. In conclusion, the endothelial cell is direct target cell of PDR's action; PDR facilitates the entry of l-arginine by serving as a carrier of l-arginine into RAEC; it also supplies aspartic acid and stimulates ASS expression, and then enhances the intracellular citrulline–NO cycle, thus increases the availability of l-arginine and NO synthesis. Therefore the effect of PDR on platelet aggregation is primarily attributed to its stimulation of NO synthesis in endothelial cells; PDR may be a better cardiovascular protective agent than l-arginine.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves1

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

In vitro effects of arginine or citrulline associated with a statine on nitrite oxide (NO) production in bovine aortic endothelial cells (BAECs)

Introduction Endothelial dysfunction occured very early in atherogenesis. It is due to decrease in NO production by endothelial cells leading to loss of vasodilation. NO is synthetised in endothelial cells by the endothelial nitric oxide synthase (eNOS) from arginine, with production of citrulline. Citrulline can be recycled in arginine, via arginino succinate synthase (ASS) and arginino succinate lyase (ASL). Moreover, statine increases eNOS activity and synthesis and therefore improves endothelial function.Materials and methods NO was determined using a nitrate and nitrite fluorimetric assay.ResultsNO production was increased in the presence of simvastatine and arginine or citrulline. This increase was more important with arginine compared to citrulline at 1mM. Without any substrate in the medium, an increase in eNOS protein level was found. In the presence of arginine 1mM, the simvastatine effect on eNOS protein level was more pronounced than in the presence of arginine 0,5mM. The effect was found also in the presence of citrulline 1mM. The same results were observed with eNOS mRNA level. Finally, when cells were incubated with arginine 0,5mM, simvastatine lead to increase ASL and ASS mRNA expression.ConclusionThe association of substrate with simvastatine induces an increase NO production, compared with arginine or citrulline or statine only, which is in agreement with the increase in BAECs of eNOS mRNA and protein levels.

A conserved sequence in the argininosuccinate synthetase 3′ untranslated region binds specifically to carbamyl phosphate synthetase (ammonia), the first enzyme in the pathway of urea synthesis

The five enzymes of urea synthesis in mammalian liver are in separate cellular compartments: the first two are in the mitochondrial matrix and the next three in the cytoplasm. The protein levels of all of the enzymes are coordinately regulated at both the transcriptional and post‐transcriptional levels. The pathway is highly ordered in situ, with the three cytoplasmic enzymes organized around the mitochondria. The mRNAs of two of the cytoplasmic enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase, are also localized around the mitochondria. The ASS 3′ untranslated region (UTR) has a conserved sequence containing motifs implicated in post‐transcriptional regulation of various mRNAs in several organisms; we therefore did RNA/protein binding studies with this UTR and rat hepatocyte cytoplasmic extracts. Deletions and mutations showed that the conserved sequence was essential for protein binding. A 144 kDa protein that bound specifically to the ASS 3′UTR was obtained by RNA affinity chromatography, and identified by tandem mass spectrometry as an N‐terminally‐cleaved form of carbamyl phosphate synthetase (ammonia) (CPS I), the first enzyme of the pathway. Tissue transglutaminase bound the UTR non‐specifically. The results strongly suggest that there may be some CPS I in the cytoplasm that is involved in the post‐transcriptional regulation of ASS mRNA. Support: NSF #MCB 9601421 &amp; 9983005.

The level of nitrogen supply modulates the expression of arginine metabolizing enzymes in Caco‐2/TC7 intestinal cells.

Portal arginine (Arg) plays a key role in liver ureagenesis. Intestine ability to control Arg availability from the diet may depend on a modulation of arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase (ASL) and synthetase (ASS) expression, according to protein supply. The aim of this work was to study the influence of amino acid (AA) supply on the intestinal expression of these enzymes.Caco‐2/TC7 cells were exposed for 3 days to different media containing AA at 0 (0), 1 (1X), 2 (2X) or 4 (4X) fold plasma post‐prandial concentrations. Enzymes mRNA were quantified using real time PCR. 18S rRNA was used as an internal control. Results (mean ± SEM) were analyzed by linear regression.There was an inverse relationship between AA supply and glutaminase (p= 0,028), OAT (p= 0,035), ASS (p= 0,028) and arginase (p= 0,058) expressions.This study shows for the first time a direct effect of AA supply on transcriptional regulation of arginine metabolizing enzymes. Such a regulation would enable in situations of low protein supply to increase Arg catabolism and to decrease hepatic AA oxidation thus promoting nitrogen sparing.

Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus

A double-stranded (ds) RNA hairpin-mediated down-regulation system was developed for the cultivated mushroom Agaricus bisporus, and the role of the urea cycle enzyme argininosuccinate lyase ( asl) in mushroom post-harvest development was investigated. Hairpin expression vectors were constructed to initiate down-regulation of asl and introduced into A. bisporus by Agrobacterium tumefaciens-mediated transformation. Transcripts of asl were significantly reduced (93.1 and 99.9 %) in two transformants and hairpin vector transgene sequences were maintained throughout sporophore development. Single and multiple hairpin integration events were observed in Southern analysis. Transformants with down-regulated asl exhibited reduced yield and cap expansion during post-harvest sporophore development. There were no detectable differences in urea levels between the hairpin-transformed and control strains. This is the first report of reduced gene expression resulting from the introduction of dsRNA hairpins in A. bisporus and the applications of this technology will facilitate functional studies in the mushroom.

Study of serum argininosuccinate lyase determination for diagnosis of liver diseases

The objectives of this research were to establish an automatic analysis method for the determination of serum argininosuccinate lyase (ASL) and to investigate the value of serum ASL test in the diagnosis of various liver disorders. According to the chemical reaction catalyzed by ASL, an enzyme-coupled reaction system was designed, and a methodology evaluation of this method was performed. A total of 291 patients with various liver diseases, 247 patients with nonliver disease and 32 healthy controls, were recruited, their serum levels of ASL and traditional hepatopathy markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and total bilirubin (TBil), were all determined, and their diagnostic values in liver diseases were analyzed and compared. Liver biopsy and the score of histopathological inflammation grading were performed in 31 patients with hepatopathy to explore the correlation between serum ASL level and hepatic histopathological change. A continuous monitoring assay method of serum ASL activity was established, which could be performed with automatic biochemistry analyzer. Methodological evaluation exhibited that the precision of this method was good indicated by the 4.0% intraassay coefficient of variation (CV), and 5.9% interassay CV. The mean recovery was 100.5%, linear range was from 0 to 167.7 U/L, and the lowest detection limit was approximately 0 U/L. All of the tested hepatopathy markers listed above were significantly increased in the liver disease group. However, levels of traditional markers of hepatopathy were all significantly increased at different degrees (all P<0.001) in patients with nonliver diseases; in contrast, there were no significantly increased ASL levels in all non-hepatopathy groups (P=0.335). The receiver operating characteristic (ROC) curve showed that the sensitivity and specificity of ASL were 100% and 91.1% (cutoff value=8 U/L), respectively, in the assessment of liver diseases. In contrast, ALT levels were 97.6% and 24.7%, and AST levels were 83.8% and 28.3% (both cutoff values=40.0 U/L), respectively. A positive correlation (r=0.417, P=0.019) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (SHIG) (9.83+/-3.36). The sensitivity and specificity of ALS is much higher than that of ALT and AST for the diagnosis of liver diseases. ASL may be a more valuable marker for estimating hepatopathy.

Native Umbilical Cord Matrix Stem Cells Express Hepatic Markers and Differentiate Into Hepatocyte-like Cells

Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

L-Arginine metabolism in dog kidney and isolated nephron segments

The renal basic amino acid metabolism often differs in rodents, strict carnivores, and omnivore species. Given the pivotal role of l-arginine and l-ornithine in several metabolic pathways and the fact that the dog is closely related to humans, being also an omnivore, we tested whether l-arginine metabolism and l-ornithine catabolism take place in the dog kidney. We examined the metabolism of l-arginine in dog cortical tubules to integrate local l-arginine metabolism into a general physiological and metabolic framework. To achieve these goals, we first ascertained the protein expression of relevant enzymes by Western blot. l-Arginine catabolism was studied in suspensions of canine cortical proximal tubules, medullary thick ascending limbs, and papillary collecting ducts either incubated without exogenous l-arginine being added (small endogenous quantities) or incubated with l-arginine being added in supraphysiological amounts (2 mmol/L with or without the presence of alternative metabolic substrates, 2 mmol/L l-glutamine, or lactate). The results revealed that dog kidneys consumed l-citrulline and released l-arginine and l-ornithine. Argininosuccinate synthetase and lyase, arginase II, and ornithine aminotransferase were detected in the renal cortex. Arginase II activity was found in a suspension of proximal tubules by measuring the amounts of urea and l-ornithine produced. A fraction of this l-ornithine was further partially metabolized through the intramitochondrial ornithine aminotransferase pathway, leading to changes in l-glutamate, glucose, l-alanine, and ammonia metabolism without l-proline accumulation. Medullary thick ascending limbs expressed a very low arginase activity, whereas papillary collecting ducts did not. In conclusion, the dog kidney produces l-arginine. Part of this l-arginine is further catabolized by arginase II, suggesting that its physiological role was to produce l-ornithine for the body.

Usefulness of Magnetic Resonance Spectroscopy in Urea Cycle Disorders

Late-onset urea cycle disorders are characterized by chronic encephalopathy and a risk of hyperammonemic crises triggered by environmental stressors. Reported here is the case of a 30-year-old woman with chronic encephalopathy due to argininosuccinate lyase deficiency. Cerebral proton magnetic resonance spectroscopy showed brain creatine deficiency and its normalization during treatment. These findings are in keeping with recent reports suggesting that creatine deficiency is involved in the neurological dysfunction of urea cycle disorders and demonstrate the usefulness of magnetic resonance spectroscopy in the diagnosis and follow-up of urea cycle disorders.

Activated microglia cells express argininosuccinate synthetase and argininosuccinate lyase in the rat brain after transient ischemia

Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO- l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS.

Interference with the citrulline‐based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts

There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of L-citrulline from L-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 microM), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.

Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) Point Mutation by Recombination with a Truncated Nonfunctional ARG7 Gene

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.

Hyperammonemia Due to Valproic Acid in the Psychiatric Setting

Hyperammonemia Due to Valproic Acid in the Psychiatric Setting