Argininosuccinate Lyase Activity Research Articles

Effects of dietary D-mannose supplementation on growth performance, intestinal digestive capacity, gut microbiota, and ammonia tolerance of largemouth bass Micropterus salmoides

To investigate the effects of D-mannose on the growth performance, intestinal digestion capacity, intestinal microbiota composition, and ammonia tolerance of largemouth bass, experiments were conducted using largemouth bass fry with an average body weight of 7.41±0.05 g. Three experimental groups were designed, with 3 replicates per group and 30 fish per replicate. The fish were fed with feed containing different concentrations of D-mannose (0.00%, 1.00%, and 2.00% - Control, 1% mannose, 2% mannose) and were given full satiation twice a day for 56 days. The results indicated that the addition of 2% D-mannose in the feed enhanced the growth performance of the largemouth bass while reducing the feed conversion ratio. The addition of D-mannose in the feed increased the activity of digestive enzymes in the intestines (lipase, α-amylase, pepsin, alkaline phosphatase, Na+/K+ATPase, gamma-glutamyl transpeptidase, and creatine kinase). It also led to an increase in intestinal villi height and intestinal wall thickness; Adding D-mannose reduced the abundance of Staphylococcus and Achromobacter in the gut microbiota, while increasing the abundance of Aurantimicrobium. After being subjected to ammonia stress for 96 hours, the addition of D-mannose in the feed resulted in a decrease in mortality rate and neuronal nitric oxide synthase activity, while simultaneously enhancing the activity of liver argininosuccinate synthetase, argininosuccinate lyase, arginase, ornithine transcarboxylase, and inducible nitric oxide synthase. Adding D-mannose to the feed promotes the growth of largemouth bass, enhances their digestive capacity, and has a positive impact on their intestinal microbiota composition. Furthermore, it has been demonstrated that it can enhance the tolerance of largemouth bass to ammonia.

Zinc Imidazole Framework‐8 Nanoparticles Disperse MRSA Biofilm by Inhibiting Arginine Biosynthesis and Down‐Regulating Adhesion‐Related Proteins

AbstractNanomaterials, including ZIF‐8 nanoparticles (NPs), are shown to be effective antimicrobial agents against Methicillin‐resistant Staphylococcus aureus(MRSA). However, the antibiofilm properties and mechanisms of ZIF‐8 NPs remain uncertain. In this study, ZIF‐8 NPs are prepared using the room temperature solution reaction method and characterized. Biofilm formation inhibition test and biofilm eradication test are performed and the results show that ZIF‐8 NPs can inhibit the formation of MRSA biofilm as well as disperse established MRSA biofilm. Proteomics and real‐time fluorescence quantitative polymerase chain reaction (PCR) are conducted to prove that ZIF‐8 NPs reduce the expression of adhesion‐related proteins, namely the fibronectin‐binding proteins A and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastin binding protein (ebps), and fibrin binding protein (eno). ZIF‐8 NPs also inhibit the arginine biosynthesis pathway by affecting the activities of argininosuccinate lyase, ornithine carbamyl transferase, Glutamate dehydrogenase, carbamate kinase, and arginine deiminase. A conclusion can be drawn from the above results that ZIF‐8 NPs can inhibit bacterial adhesion and kill bacteria directly, ultimately destroying MRSA biofilm. This study provides a molecular basis for the treatment of MRSA biofilm with ZIF‐8 NPs.

Cortisol enhances citrulline synthesis from proline in enterocytes of suckling piglets.

There are marked decreases in plasma concentrations of cortisol and arginine (an essential amino acid for neonates) as well as intestinal citrulline synthesis in piglets during the first 14days of life. The objective of this study was to test the hypothesis that increasing plasma cortisol concentrations by cortisol administration may prevent the decline in intestinal citrulline and arginine synthesis from proline, thereby possibly increasing plasma arginine concentration in suckling piglets and their growth. Seven-day-old pigs reared by sows received daily intramuscular injections of hydrocortisone 21-acetate (25mg/kg) or vehicle solution (saline) (n = 10/group). At 14days of age, piglets were used to prepare jejunal enterocytes. Cells were incubated at 37°C for 30min in oxygenated Krebs buffer containing 5mM glucose, 2mM [U-14C]proline, and 2mM glutamine. Cortisol treatment increased plasma cortisol concentration, mitochondrial proline oxidase and N-acetylglutamate synthase activities, cytosolic argininosuccinate lyase activity, and the intracellular concentrations of N-acetylglutamate and carbamoyl phosphate for citrulline and arginine synthesis. However, cortisol treatment induced the expression of intestinal arginase-II for arginine hydrolysis, resulting in no change in plasma arginine concentration. Administration of cortisol had no effect on milk consumption or the whole-body growth rate of piglets, but increased villus height in the jejunum and ileum. Collectively, these results suggest an important role for proline oxidase and N-acetylglutamate in regulating citrulline and arginine synthesis from proline in pig enterocytes. Because proline catabolism plays an important role in modulating protein synthesis, cell proliferation, and arginine production, our findings may have important implications for understanding the role of proline oxidase in the growth and health of the mammalian small intestine.

Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Developmental changes in the utilization of citrulline by neonatal pigs.

Developmental changes in the renal expression and activity of argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL), enzymes that use citrulline for the production of arginine, have been reported. Thus, the ability of neonates, and especially premature neonates, to produce arginine may be compromised. To determine the utilization of citrulline in vivo, we measured renal expression of ASS1 and ASL and conducted citrulline compartmental and noncompartmental kinetics using [15N]citrulline in pigs of five different ages (from 10 days preterm to 5 wk of age). The tracer was given in substrate amounts to also test the ability of neonatal pigs to use exogenous citrulline. Preterm and term pigs at birth had lower ASS1 and ASL expression than older animals, which was reflected in the longer half-life of citrulline in the neonatal groups. The production and utilization of citrulline by 1-wk-old pigs was greater than in pigs of other ages, including 5-wk-old animals. Plasma citrulline concentration was not able to capture these differences in citrulline production and utilization. In conclusion, the developmental changes in renal ASS1 and ASL gene expression are reflected in the ability of the pigs to use citrulline. However, it seems that there is an excess capacity to use citrulline at all ages, including during prematurity, since the bolus dose of tracer did not result in an increase in endogenous citrulline. Our results support the idea that citrulline supplementation in neonatal, including premature, pigs is a viable option to increase arginine availability.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria

Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA.

Argininosuccinate lyase interacts with cyclin A2 in cytoplasm and modulates growth of liver tumor cells

Arginine is a critical amino acid in specific cancer types including hepatocellular carcinoma (HCC) and melanoma. Novel molecular mechanisms and therapeutic targets in arginine metabolism-mediated cancer formation await further identification. Our laboratory has previously demonstrated that arginine metabolic enzyme argininosuccinate lyase (ASL) promoted HCC formation in part via maintenance of cyclin A2 protein expression and arginine production for channeling to nitric oxide synthase. In this study, we investigated the mechanism by which ASL regulates cyclin A2 expression. We found that ASL interacted with cyclin A2 in HCC cells and the localization of their interaction was in the cytoplasm. Mutation of essential residues for enzymatic activity of ASL did not affect the binding of ASL to cyclin A2. Moreover, the mutant ASL retained the ability to restore the decreased tumorigenicity caused by ASL shRNA. Furthermore, overexpression of ASL conferred resistance to arginine deprivation therapy. Finally, the important pathways and potential therapeutic targets in ASL-regulated HCC were identified by bioinformatics analyses with Metacore database and Connectivity Map database. Our analyses suggested that bisoprolol, celecoxib, and ipratropium bromide, are potential therapeutics for ASL-regulated HCC formation. Thus, ASL interacts with cyclin A2 in cytoplasm, and may promote HCC formation through this non-enzymatic function. Overexpression of ASL may be a contributing factor in drug resistance for arginine deprivation therapy.

Effect of Cysteamine on Mutant ASL Proteins with Cysteine for Arginine Substitutions

Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins. We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle. In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay. Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue. Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria.

Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3% of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16% of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30% of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.

Normal root elongation requires arginine produced by argininosuccinate lyase in rice

Plant roots play an important role in the uptake of water and nutrients, structural support and environmental sensing, but the molecular mechanisms involved in root development are poorly understood in rice (Oryza sativa), which is characterized by a dense fibrous root system. Here we report a rice mutant (red1 for root elongation defect 1) with short roots. Morphological and physiological analyses showed that the mutant had a shorter length from the quiescent center (QC) to the starting point of the elongation zone but a similar cell size and number of lateral and crown roots compared with the wild type. Furthermore, the mutant had similar radial structure and nutrient uptake patterns to the wild type. Map-based cloning revealed that the mutant phenotype was caused by a point mutation of a gene encoding an argininosuccinate lyase (ASL), catalyzing the last step of arginine biosynthesis. The OsASL1 gene has two distinct transcripts, OsASL1.1 and OsASL1.2, which result from different transcription start sites, but only OsASL1.1 was able to complement the mutant phenotype. OsASL1.1 was expressed in both the roots and shoots. The protein encoded by OsASL1.1 showed ASL activity in yeast. OsALS1.1 was localized to the plastid. The short root of the mutant was rescued by exogenous addition of arginine, but not by other amino acids. These results indicate that arginine produced by ASL is required for normal root elongation in rice.

Citrulline uptake in rat cerebral cortex slices: Modulation by Thioacetamide -Induced hepatic failure

L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [3H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF (“TAA slices”). In both control and TAA slices the uptake was partially Na+-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y+/y+L transport system substrate, nor by amino acids taken up by systems A, xc −or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.

Understanding the Role of Argininosuccinate Lyase Transcript Variants in the Clinical and Biochemical Variability of the Urea Cycle Disorder Argininosuccinic Aciduria

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells

BackgroundLoss of function of fumarate hydratase (FH), the mitochondrial tumor suppressor and tricarboxylic acid (TCA) cycle enzyme, is associated with a highly malignant form of papillary and collecting duct renal cell cancer. The accumulation of fumarate in these cells has been linked to the tumorigenic process. However, little is known about the overall effects of the loss of FH on cellular metabolism.MethodsWe performed comprehensive metabolomic analyses of urine from Fh1-deficient mice and stable isotopologue tracing from human and mouse FH-deficient cell lines to investigate the biochemical signature of the loss of FH.ResultsThe metabolomics analysis revealed that the urea cycle metabolite argininosuccinate is a common metabolic biomarker of FH deficiency. Argininosuccinate was found to be produced from arginine and fumarate by the reverse activity of the urea cycle enzyme argininosuccinate lyase (ASL), making these cells auxotrophic for arginine. Depleting arginine from the growth media by the addition of pegylated arginine deiminase (ADI-PEG 20) decreased the production of argininosuccinate in FH-deficient cells and reduced cell survival and proliferation.ConclusionsThese results unravel a previously unidentified correlation between fumarate accumulation and the urea cycle enzyme ASL in FH-deficient cells. The finding that FH-deficient cells become auxotrophic for arginine opens a new therapeutic perspective for the cure of hereditary leiomyomatosis and renal cell cancer (HLRCC).

α-Crystallin protects human arginosuccinate lyase activity under freeze–thaw conditions

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in‐vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme. Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299T > C), p.V178M (c.532G > A), p.E189G (c.566A > G), p.Q286R (c.857A > G), p.K315E (c.943A > G), p.R379C (c.1135C > T) and p.R385C (c.1153C > T) in comparison to the wildtype protein. In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K(m) values for argininosuccinic acid differed significantly from the wild-type ASL protein. The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.

Decreased glutamine synthetase, increased citrulline–nitric oxide cycle activities, and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2h and the long term (chronic) group animals were killed after 5days of single injection of KA (15mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.

Abstract 4848: Pegylated arginine deiminase induces autophagy in canine melanoma and canine osteosarcoma

Abstract Arginine is a nonessential amino acid in adult mice, men and dogs, which undergoes both synthesis and degradation in complex series of compartmentalized steps at the cellular and systemic level. The urea cycle enzymes are responsible for this axis and is dependent on the levels of are argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL). It has been shown that many human tumor types are auxotrophic for L-arginine typically due to very low or no specific activities of ASS1 and/or ASL and are thus sensitive to induction of cell death as a result of the depravation of this single nutrient. Pegylated arginine-deiminase (PEGADI) is a molecule with this mechanism of action and has entered phase I and II clinical trials in humans where it has proven its clinical utility in the treatment of hepatocellular carcinoma and melanoma. The cancer-bearing dog serves an excellent spontaneous out-bred immune competent large animal model for numerous homologous neoplastic diseases in humans. The purpose of this study is to determine the status of arginine-synthetic enzymes and then evaluate the action of PEGADI in cultured canine cancer melanoma (12, 23, 36) and osteosarcoma (D17, 48-4, 29) cell lines. Using western blot analysis we found that each of these six canine cell lines expressed ASS1 and ASL. PEGADI depleted arginine in a time- and concentration-dependent manner in the cultured canine melanoma and osteosarcoma as measured by HPLC. Furthermore, the canine melanoma and osteosarcoma cells were very sensitive to this depletion. In particular, there was a dramatic loss of clonogenic survival in these cells treated with PEGADI (IC50 range: 0.03 uM at 96 h to 0.3 at 24 h). This sensitivity was apparently due to caspase independent cell killing as caspase 3, 8, or 9 were not activated. Changes in cell cycle distribution were quantitated with flow cytometry. Despite the lack of caspase activation, there was an increase in the number of cells in the Sub G1 population. To determine if PEGADI induced autophagy in these cells the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form LC3B-II was assessed and showed there was a dose dependent induction of LC3B-II. Consistent with the induction of autophagy, results demonstrate an induction in the formation autophagolysosomes as determined by flow cytometery analysis of acridine orange staining of acidic vesicles, as well as fluorescent microscopy of treated cells stained with monodansylcadaverine. Our results support the notion that PEGADI may have clinical utility in the management of canine neoplasia. A clinical trial for this species is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4848.

Long-term outcome of patients with argininosuccinate lyase deficiency diagnosed by newborn screening in Austria

Twenty-three patients with late onset argininosuccinate lyase deficiency (ASLD) were identified during a 27-year period of newborn screening in Austria (1:95,600, 95% CI=1:68,036–1:162,531). One additional patient was identified outside the newborn screening with neonatal hyperammonemia. Long-term outcome data were available in 17 patients (median age 13years) ascertained by newborn screening. Patients were treated with protein restricted diet and oral arginine supplementation during infancy and childhood. IQ was average/above average in 11 (65%), low average in 5 (29%), and in the mild intellectual disability range in 1 (6%) patients. Four patients had an abnormal EEG without evidence of clinical seizures and three had abnormal liver function tests and/or evidence of hepatic steatosis. Plasma citrulline levels were elevated in four patients. Plasma ammonia levels were within normal range prior and after a protein load in all patients. Seven different mutations were identified in the 16 alleles investigated. Four mutations were novel (p.E189G, p.R168C, p.R126P, and p.D423H). All mutations were associated with low argininosuccinate lyase activities (0–15%) in red blood cells. Newborn screening might be beneficial in the prevention of chronic neurologic and intellectual sequelae in late onset ASLD, but a proportion of benign variants might have contributed to the overall favorable outcome as well.