Argininosuccinate Synthase Expression Research Articles

Inhibition of argininosuccinate synthase (ASS) affected ammonia excretion in yellow catfish Pelteobagrus fulvidraco during acute ammonia poisoning

Ammonia is toxic to most fish. Different fish have different treatment methods for ammonia toxicity, mainly including urea cycle and glutamine synthesis pathway, depending on their physiological status. A two-stage study was carried out to test the inhibition of argininosuccinate synthase (ASS) effects on ammonia detoxification in hyperammonemia yellow catfish. At the stage 1, fish were injected lethal half concentration (LC50) of ammonium acetate and sampled at 0, 1, 3, 6, 12 and 24 h post-injection. The result found that plasma ammonia content of fish was injected with ammonium acetate reached its highest level at hour 1, which was used as the optimal sampling time in stage 2. At the stage 2, the four experimental groups were carried out: NaCl group was injected intraperitoneally with 0.9% sodium chloride as control; NH3 group was injected with LC50 ammonium acetate; NH3+BPP group was injected with mixture of ammonium acetate and bradykinin-potentiating peptide (BPP); BPP group was injected with BPP. The results showed that inhibition of ASS can lead to accumulation of ammonia in plasma and kidney, and glutamine in liver and kidney; and decreases ASS, argininosuccinate lyase, arginase activates, and ornithine, glutamate, alanine, proline, tyrosine, methionine contents, and increases ornithine transcarbamylase, glutaminase activates, and citrulline, aspartate, arginine, glutamine, histidine, isoleucine, phenylalanine, lysine contents; and result in upregulation of ASS, ASL, CPS 1, OTC, GS and GLS genes expression. This result indicates that the inhibition of ASS in yellow catfish with ammonia poisoning will lead to the urea cycle disorder; yellow catfish can enhance the detoxification capacity of ammonia by improving glutamine synthesis when the urea cycle is inhibited; argininase played an important role in ammonia detoxification of yellow catfish.

Effects of Thermal Conditioning on Changes in Hepatic and Muscular Tissue Associated With Reduced Heat Production and Body Temperature in Young Chickens.

Effects of increased summer temperatures on poultry production are becoming more pronounced due to global warming, so it is important to consider approaches that might reduce heat stress in chickens. Thermal conditioning in chickens in the neonatal period can improve thermotolerance and reduce body temperature increases when birds are exposed to high ambient temperature later in life. The objective of this study was to investigate physiological and molecular changes associated with heat production and hence body temperature regulation under high ambient temperatures in thermally conditioned chicks. Three-day-old broiler chicks (Chunky) were thermally conditioned by exposure to a high ambient temperature (40°C) for 12 h while control chicks were kept at 30°C. Four days after the treatment, both groups were exposed to 40°C for 15 or 90 min. The increase in rectal temperature during 90 min of exposure to a high ambient temperature was less in thermally conditioned than control chicks. At 15-min of re-exposure treatment, gene expression for uncoupling protein and carnitine palmitoyletransferase 1, key molecules in thermogenesis and fatty acid oxidation, were significantly higher in pectoral muscle of control chicks but not conditioned chicks. Hepatic argininosuccinate synthase (ASS) decreased and hepatic argininosuccinate lyase (ASL) increased after reexposure to a high temperature. The concentrations of hepatic arginosuccinic acid, and ASS and ASL expression, were upregulated in conditioned chicks compared with the control chicks, indicating activity of the urea cycle could be enhanced to trap more energy to reduce heat production in conditioned chicks. These results suggest thermal conditioning can reduce the increase in heat production in muscles of chickens that occurs in high ambient temperatures to promote sensible heat loss. Conditioning may also promote energy trapping process in the liver by altering the heat production system, resulting in an alleviation of the excessive rise of body temperature.

Protective effects of distinct proline-rich oligopeptides from B. jararaca snake venom against oxidative stress-induced neurotoxicity

The low molecular mass fraction from Bothrops jararaca snake venom contains proline-rich peptides (Bj-PROs), also known as bradykinin-potentiating peptides (BPPs), which were described as neuroprotective against H2O2-induced oxidative stress. Recently, we reported that the neuroprotective action of Bj-PRO-10c (<ENWPHPQIPP) is a result of an increase in the expression and activity of argininosuccinate synthase (AsS), thereby improving increase L-arginine synthesis. It is proposed that formation agmatine and polyamines from L-arginine metabolism are neuroprotective, reducing lipid peroxidation, oxidative stress, and maintenance of the mitochondrial membrane. In the present study, Bj-PRO-5a (<EKWAP), Bj-PRO-7a (<EDGPIPP), Bj-PRO-11e (<EARPPHPPIPP), and Bj-PRO-AP (<EARPPHPPIPPAP) – with different structure-activities, in particular on AsS expression and activity – were selected to study their protective properties on H2O2-induced oxidative stress in SH-SY5Y cells. Bj-PROs tested were neuroprotective, but Bj-PRO-5a and Bj-PRO-7a increased cell viability by more than 85% in the presence of H2O2 (450 μM) and reduced the oxidative stress markers. AsS, iNOS, and NF-ĸB expressions were not involved in the neuroprotective mechanism for both peptides, in contrast to Bj-PRO-10c, as reported in the literature. Bj-PRO-5a is a neuroprotector peptide that decreases ROS production, NO levels lipid peroxidation, and changes in mitochondrial membrane permeability caused by H2O2 exposition. The neuroprotective property of Bj-PRO-7a against H2O2 is also related to decreased ROS production and lipid peroxidation but without altering NO levels. These results show that the protective effects of different Bj-PROs against oxidative stress can be explained by distinct mechanisms independent of an increase in L-arginine bioavailability arising from AsS activity. Further studies are now needed to reveal the details of the neuroprotective mechanisms exerted by Bj-PROs.

Serendipita indica modulates expression of arginine metabolism genes during colonization of Arabidopsis thaliana roots

Abstract Endophytic fungus Serendipita indica belongs to the order Sebacinales. It is an extremely versatile root endophyte colonizing more than 150 different plant species including the model plant Arabidopsis thaliana. Colonization of S. indica triggers enhanced growth, early flowering, increase in seed content, alteration of secondary metabolite pathways and adaption to different biotic and abiotic stresses. This is why this fungus got ample attention by the plant researchers. In this study, we report modulation in expression of argininosuccinate synthase (AS), argininosuccinate lyase (AL) and arginase 2 (ARGAH2) genes in A. thaliana as a result of S. indica colonization. Similarly, expression of GUS reporter gene driven by AS and ARGAH2 promoters in roots was altered. AS, AL and ARGAH2 genes were down-regulated at early stage of root colonization i.e at 3 days post inoculation (dpi), with a subsequent up-regulation at later stages (7 and 14 dpi). Other arginine catabolism genes, Arginine decarboxylase-1 (ADC1), Arginine decarboxylase-2 (ADC2) and Pyrroline-5-carboxylate reductase (P5CR) were strongly up-regulated at later stages of S. indica colonization. Our findings show significant changes in expression of several important genes in arginine metabolic pathway upon S. indica colonization. These changes are very specific and strongly depend on the colonization phase of the fungus. Keywords: AL; ARGAH1; ARGAH2; AS; Arginine metabolism; GUS; Quantitative RT-PCR; Serendipita indica http://dx.doi.org/10.19045/bspab.2018.700203

Bradykinin-potentiating PEPTIDE-10C, an argininosuccinate synthetase activator, protects against H2O2-induced oxidative stress in SH-SY5Y neuroblastoma cells

Bradykinin-potentiating peptides (BPPs – 5a, 7a, 9a, 10c, 11e, and 12b) of Bothrops jararaca (Bj) were described as argininosuccinate synthase (AsS) activators, improving l-arginine availability. Agmatine and polyamines, which are l-arginine metabolism products, have neuroprotective properties. Here, we investigated the neuroprotective effects of low molecular mass fraction from Bj venom (LMMF) and two synthetic BPPs (BPP-10c, <ENWPHPQIPP; BPP-12b, <EWGRPPGPPIPP) in the SH-SY5Y cell line against H2O2-induced oxidative stress. The neuroprotective effects against H2O2-induced were analyzed by reactive oxygen species (ROS – DCFH) production; lipid peroxidation (TBARS); intracellular GSH; AsS, iNOS, and NF-kB expressions; nitrite levels (Griess); mitochondrial membrane potential (TMRM); and antioxidant activity (DPPH). Analysis of variance followed by Tukey’s post hoc test were calculated for statistical comparisons. Pre-treatment with both BPPs significantly reduced cell death induced by H2O2, but BPP-10c showed higher protective capacity than BPP-12b. LMMF pretreatment was unable to prevent the reduction of cell viability caused by H2O2. The neuroprotective mechanism of BPP-10c against oxidative stress was investigated. BPP-10c reduced ROS generation and lipid peroxidation in relation to cells treated only with H2O2. BBP-10c increased AsS expression and was not neuroprotective in the presence of MDLA, a specific inhibitor of AsS. BPP-10c reduced iNOS expression and nitrate levels but decreased NF-kB expression. Furthermore, BPP-10c protected the mitochondrial membrane against oxidation. Overall, we demonstrated for the first time neuroprotective mechanisms of BPPs against oxidative stress, opening new perspectives to the study and application of these peptides for the treatment of neurodegenerative diseases.

Altered Expression of Urea Cycle Enzymes in Amyloid-β Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

Reconstitution of T Cell Proliferation under Arginine Limitation: Activated Human T Cells Take Up Citrulline via L-Type Amino Acid Transporter 1 and Use It to Regenerate Arginine after Induction of Argininosuccinate Synthase Expression.

In the tumor microenvironment, arginine is metabolized by arginase-expressing myeloid cells. This arginine depletion profoundly inhibits T cell functions and is crucially involved in tumor-induced immunosuppression. Reconstitution of adaptive immune functions in the context of arginase-mediated tumor immune escape is a promising therapeutic strategy to boost the immunological antitumor response. Arginine can be recycled in certain mammalian tissues from citrulline via argininosuccinate (ASA) in a two-step enzymatic process involving the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Here, we demonstrate that anti-CD3/anti-CD28-activated human primary CD4+ and CD8+ T cells upregulate ASS expression in response to low extracellular arginine concentrations, while ASL is expressed constitutively. ASS expression peaked under moderate arginine restriction (20 µM), but no relevant induction was detectable in the complete absence of extracellular arginine. The upregulated ASS correlated with a reconstitution of T cell proliferation upon supplementation of citrulline, while the suppressed production of IFN-γ was refractory to citrulline substitution. In contrast, ASA reconstituted proliferation and cytokine synthesis even in the complete absence of arginine. By direct quantification of intracellular metabolites we show that activated primary human T cells import citrulline but only metabolize it further to ASA and arginine when ASS is expressed in the context of low amounts of extracellular arginine. We then clarify that citrulline transport is largely mediated by the L-type amino acid transporter 1 (LAT1), induced upon human T cell activation. Upon siRNA-mediated knockdown of LAT1, activated T cells lost the ability to import citrulline. These data underline the potential of citrulline substitution as a promising pharmacological way to treat immunosuppression in settings of arginine deprivation.

Diminished Citrulline‐Arginine‐Nitric Oxide Production Rates are Associated with Necrotizing Enterocolitis Incidence in Premature Pigs

Necrotizing enterocolitis (NEC) is a major gastrointestinal disease in premature infants that is associated with formula feeding and intestinal hypoxia. Low arginine availability in these infants has been linked to NEC since arginine is the sole precursor of nitric oxide (NO), a critical mediator of vasodilation, blood flow and tissue oxygenation. Arginine availability is dependent on its de novo production from gut‐derived citrulline and the action of the rate‐limiting enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) in the gut and other tissues. However, arginine availability is balanced by arginine catabolism to either NO by NO synthase 2&amp;3 (NOS2&amp;3) or to ornithine by arginase 1&amp;2 (Arg1&amp;2). We hypothesized that citrulline‐arginine‐NO production rates are limiting in premature pigs, which predisposes them to NEC. Cesarean‐derived preterm (90% term) and term (98–100% term) pigs were provided TPN for 24 h, followed by enteral formula for 48 h to induce NEC. Intravenous infusions of [13C6]arginine, [15N‐ureido]citrulline and [15N18O3]nitrate were provided throughout feeding to quantify in vivo citrulline‐arginine‐NO kinetics. Preterm and term pig tissues were also collected at birth, and we report that intestinal expression of the arginine producing genes ASS and ASL were lower in preterm vs term pigs (P&lt;0.05). However, the expression of arginine catabolizing genes NOS2&amp;3 and Arg1&amp;2 in gut were not affected by gestational age at birth. Intestinal gene expression was further assessed after formula feeding, and Arg2 and NOS3 expression were increased in preterm vs term pigs, while ASS expression remained low in preterm pigs after feeding (P&lt;0.05). Histological scoring of fed pigs showed that NEC incidence was 90% in preterm pigs, whereas term pigs did not get NEC. Importantly, the rates of arginine‐citrulline‐NO flux showed that preterm pigs produced ~40–50% less citrulline, arginine and NO than term pigs regardless of feeding mode (P&lt;0.05). To better assess NO metabolism we also quantified its oxidation products, nitrate and nitrite. Nitrate flux was ~60% lower in preterm pigs during TPN (P&lt;0.05), and all pigs exhibited a drastic reduction in nitrate flux after the introduction of enteral formula. Strikingly, there was a marked supraphysiological nitrite surge in plasma prior to and immediately after oral feed initiation in preterm piglets that was absent in term pigs. Moreover, preterm pigs with higher post‐feeding plasma nitrite surges were NEC‐resistant (P&lt;0.05). Our results suggest that depressed intestinal blood flow and oxygenation that are implicated in NEC may be linked to the diminished capacity to produce citrulline‐arginine‐NO necessary for vasodilation. These findings provide a rationale for arginine supplementation of preterm infants to overcome the limited capacity for arginine and NO production that predisposes NEC etiology.Support or Funding InformationGrant Funding Source: USDA/ARS

Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells

Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20μg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction.

Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer&#039;s disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aβ precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD.

Ferulic acid enhances nitric oxide production through up-regulation of argininosuccinate synthase in inflammatory human endothelial cells

AimIn this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-α-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved. Main methodsThe TNF-α-stimulated inflammatory HUVECs were treated with acetylcholine (ACh) and/or FA. NO productions were measured by monitoring nitrite and nitrate using a 2,3-diaminonaphthalene Kit. Expressions of mRNA and proteins were evaluated by RT-PCR and Western blotting, respectively. Key findingsFA treatment resulted in a dose-dependent (10–200μM) restoration of ACh-mediated NO production in TNF-α-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O2− production in TNF-α-stimulated HUVECs. NG-monomethyl-l-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and α-methyl-dl-aspartic acid (an argininosuccinate synthase (ASS) inhibitor) counteracted the effects of FA on the NO production. While FA treatment did not significantly affect the protein expression of p-eNOS or eNOS, the protein expression of ASS as well as mRNA expression was restored to normal levels upon exposure to FA in TNF-α-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-κB) expression by TNF-α. SignificanceFA treatment rescues the defect in ACh-induced NO production resulting from TNF-α-stimulation in inflammatory HUVECs. This effect was likely due, in part, to the FA-mediated up-regulation of ASS expression via the suppression of NF-κB inflammatory signaling cascade.

Women with cervical cancer have high levels of citrulline that are inversely correlated with percentages of circulating IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells

Women with cervical cancer have high levels of citrulline that are inversely correlated with percentages of circulating IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells

Pegylated Arginine Deiminase (ADI-PEG20) in Relapsed/Refractory and/or Elder Acute Myeloid Leukemia: The Preliminary Result of Phase II Trial

Background: Deprivation of circulating L-asparagine by L-asparaginase, which can lead to the inhibition of RNA and DNA synthesis and subsequent apoptosis of blastic cell, has been implemented as part of multidrug chemotherapy for acute lymphoblastic leukemia since decades ago. Arginine, a semi-essential amino acid in human, is involved in diverse aspects of tumor metabolism and plays critical role for the growth of human cancers. Deficiency of argininosuccinate synthase (ASS), the rate-limiting enzyme for endogenous arginine production in urea cycle, has been found in various cancer tissues. In preclinical studies, pegylated arginine deiminase (ADI-PEG20), which can rapidly convert arginine into citrulline and serve as an arginine depriving agent, was shown to exert in vitro and in vivo anti-proliferative effect on ASS-deficient cancers, such as hepatocellular carcinoma, melanoma, small cell lung cancer, lymphoma and acute myeloid leukemia (AML). The efficacy of ADI-PEG20 is currently under evaluation for various solid tumors in clinical trial setting, including a global phase III trial for hepatocellular carcinoma. Absence of ASS expression has been noted in 87% (46/53) of bone marrow biopsy samples of patients with AML.1 In xenograft model, ADI-PEG20 could reduce the leukemic burden in mice transplanted with primary AML cells.2Herein, we reported the preliminary result of a phase II trial evaluating the therapeutic efficacy of ADI-PEG20 in relapsed/refractory and/or elderly AML patients.Patients and Methods: Patients ≥ 18 years with ASS deficient (by western blotting of bone marrow leukemia cells and/or immunohistochemical staining of bone marrow biopsy), relapsed/refractory or poor-risk AML were eligible. The poor-risk AML includes treatment-related AML, antecedent hematologic disease, unfavorable cytogenetics and de novo AML ≥ 70 years of age. Patients received ADI-PEG20 at 320IU/m2IM weekly (4 weeks as one cycle). Bone marrow aspiration was performed at the time of enrollment, and after the first and second cycle of treatment to evaluate the response. Treatment was continued for each patient until the occurrence of disease progression, development of unacceptable toxicity, death, or withdrawal of consent for any reason. If patients achieved complete remission (CR) or CR with incomplete blood count recovery, the treatment was finished after another 4 cycles of ADI-PEG20.Results: Between October 2013 and May 2014, 9 patients were enrolled, with a male/female ratio of 5/4 and a median age of 62 years (ranged 27 to 79 years old). They were all de novo AML except for 1 with blast-transformed chronic myelomonocytic leukemia. All patients received at least one prior treatment regimen, except for two treatment-naïve elderly patients. After a mean 1.5 cycles of ADI-PEG20 treatment, 2 of 8 evaluable patients achieved CR after 3 and 1 cycles of ADI-PEG20, respectively, while 6 patients had disease progression after an average of 1 cycle of treatment. One patient was not evaluable for response due to withdrawal of consent after the first 2 doses of treatment. Two patients, who died within 2 weeks after the first dose of ADI-PEG20, were considered to have progressive disease. Of the 2 CR patients, 1 was 79 years old with chemo-naïve acute megakaryocytic leukemia (M7) and the other was 69 years old with low-dose Ara-C refractory M2. The most common treatment-related severe adverse events (AE) included grade 4 tumor lysis syndrome, grade 4 infection and treatment-related grade 4 neutropenia occurring in one patient each. The episode of grade 4 neutropenia occurred in the ADI-PEG20 responsive M7 patient. Minor AE included grade 1 hyperuricemia and skin rash in 2 and 1 patients, respectively.Conclusions: ADI-PEG20 is an effective treatment for some patients with ASS-deficient AML with minimal toxicities. Further investigation with genetic and epigenetic profiling to identify patients who will benefit from arginine deprivation therapy is warranted.References. 1.Szlosarek P, et al. Pegylated arginine deiminase (ADI-PEG 20) as a potential novel therapy for argininosuccinate synthetase-deficient acute myeloid leukemia. (AACR Abstract # 467, 2012).2. Miraki-Moud F, et al. Arginine deprivation with pegylated arginine deiminase induces death of acute myeloid leukaemia cells in vivo. Blood 2012 122:1458. DisclosuresTsai:TWD Pharmaceuticals, Inc: Honoraria.

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.

L-arginine depletion by PEG-arginase I, a new potential therapy for acute lymphoblastic leukemia.

6580 Background: Advances in therapies have resulted in an overall complete remission rate of approximately 85% for childhood acute lymphoblastic leukemia (ALL). In contrast, the overall remission rate of adults with leukemia continues to be poor, only about 40% in cases of T cell-ALL (T-ALL). Therefore, it is imperative to generate new therapies that alone or in combination with other treatments could potentially increase the percentages of complete responders or be used to treat the refractory ALL population. Our published results show that a pegylated form of human arginase I (peg-Arg I) prevented T-ALL cell proliferation in vitro and in vivo through the induction of tumor cell apoptosis. Interestingly, the anti-leukemic effects induced by peg-Arg I did not affect the anti-tumor activity of normal T cells, suggesting an anti-tumor specific effect. Our hypothesis states that peg-Arg I has an anti-tumoral effect on B-ALL and T-ALL cells in vitro and that the sensitivity of ALL cells to peg-Arg I depends on their expression of argininosuccinate synthase (ASS) and their ability to produce L-arginine de novo from citrulline. Methods: Malignant T cell proliferation was tested using nonradioactive cell proliferation yellow tretrazolium salt kit. Apoptosis studies were based on the expression of annexin V. Western blot assays were conducted to determine enzymatic expression in different cell lines. Results: The results of our in vitro experiments showed that peg-Arg I had a pro-apoptotic and anti-proliferattive effect on B-ALL cells similar to the one previously seen on T-ALL cells. These effects can be overcome in cell lines able that express ASS and therefore to produce L-arginine de novo. Conclusions: Our results suggest the role of ASS in the ALL-apoptosis induced by peg-Arg-I. Our next steps include: _Understand why ASS-expressing ALL cells do not undergo apoptosis when cultured with peg-Arg-I_Determine the role of ASS in the anti-leukemic effect induced by peg-Arg-I in vivo. Completion of this research is expected to lead to a better understanding of how peg-Arg-I kills ALL cells and could provide the foundation for a novel therapy for ALL patients.

Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats

Baroreflex sensitivity is disturbed in many people with cardiovascular diseases such as hypertension. Brain deficiency of nitric oxide (NO), which is synthesized by NO synthase (NOS) in the citrulline-NO cycle (with argininosuccinate synthase (ASS) activity being the rate-limiting step), contributes to impaired baroreflex. We recently showed that a decapeptide isolated from Bothrops jararaca snake venom, denoted Bj-PRO-10c, exerts powerful and sustained antihypertensive activity. Bj-PRO-10c promoted vasodilatation dependent on the positive modulation of ASS activity and NO production in the endothelium, and also acted on the central nervous system, inducing the release of GABA and glutamate, two important neurotransmitters in the regulation of autonomic systems. We evaluated baroreflex function using the regression line obtained by the best-fit points of measured heart rate (HR) and mean arterial pressure (MAP) data from spontaneously hypertensive rats (SHRs) treated with Bj-PRO-10c. We also investigated molecular mechanisms involved in this effect, both in vitro and in vivo. Bj-PRO-10c mediated an increase in baroreflex sensitivity and a decrease in MAP and HR. The effects exerted by the peptide include an increase in the gene expression of endothelial NOS and ASS. Bj-PRO-10c-induced NO production depended on intracellular calcium fluxes and the activation of a G(i/o)-protein-coupled metabotropic receptor. Bj-PRO-10c induced NO production and the gene expression of ASS and endothelial NOS in the brains of SHRs, thereby improving baroreflex sensitivity. Bj-PRO-10c may reveal novel approaches for treating diseases with impaired baroreflex function.

Abstract 4519: Small cell lung cancers lack expression of argininosuccinate synthase (ASS) and are sensitive to arginine deprivation using arginine deiminase-PEG20 (ADI-PEG20)

Abstract Various tumor types have been shown to lack expression of the enzyme argininosuccinate synthase (ASS) that is required for the synthesis of arginine and thus rely on exogenous sources for protein synthesis requirements. These tumors may be sensitive to arginine deprivation, and the absence of ASS expression may serve as a biomarker for this sensitivity. Arginine deiminase (ADI) is a microbial enzyme that has been shown to efficiently lower peripheral blood arginine. Its therapeutic efficacy has been previously explored in the treatment of melanoma and hepatocellular cancer in early phase clinical trials but clinical efficacy in these tumor types has been limited. However, other tumor types lacking ASS may be more sensitive to arginine depletion. We have investigated ASS expression in human small cell lung cancer (SCLC) tumor tissue by immunohistochemistry (IHC) and have so far found that about 43% (7/16) of SCLC tumors were negative for ASS. SCLC has a robust initial response to chemotherapy but the majority of cases relapse and there remain limited options in the treatment of refractory disease. Based on the IHC findings and the need for novel therapies in recurrent SCLC, we explored the effects of pegylated ADI (ADI-PEG20) mediated arginine deprivation in SCLC in vitro and in vivo. We screened a panel of 10 human SCLC cell lines for ASS mRNA and protein expression by Real Time PCR and Western blot analysis. Little or no ASS was observed in 50% (5 of 10) of the SCLC cell lines examined and this frequency is approximately equivalent to that found in the SCLC tumor tissues. In cell proliferation assays a clear correlation between expression of ASS and sensitivity to ADI-PEG20 was detected. Biochemical analysis of the mechanism of ADI-PEG20 induced cell death in ADI sensitive cell lines revealed induction of autophagy and cell death through caspase independent mechanisms. Assessment of ADI-PEG20 activity in vivo using mice bearing SCLC xenografts demonstrated that ADI-PEG20 treatment of ASS negative SCLC xenografts caused significant and dose dependent inhibition of tumor growth, while no effect was observed with placebo controls. These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this treatment in patients with ASS negative SCLC by IHC has been initiated. 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 4519.

Abstract 4847: Arginine deprivation by PEG-ADI induces autophagic cell death and enhances the tumor suppression effect of gemcitabine in pancreatic cancer

Abstract Metabolic stress has been recently recognized to induce autophagic cell death; we have recently shown that nearly 80% of pancreatic adenocarcinoma tumor specimens are auxotrophic for the semi-essential amino acid arginine due to transcriptional suppression of argininosuccinate synthase (ASS). We hypothesized that selective arginine deprivation would induce autophagy and furthermore, by targeting alternative death pathways would increase the cytotoxic response to the chemotherapy gemcitabine. Methods: The human pancreatic cell line MIA-PaCa-2 was shown to be deficient in ASS by western blot. Arginine deprivation was achieved by treatment of cells and xenografts with PEG-ADI, which shuttles arginine to citrulline. Cells were treated with either PEG-ADI, gemcitabine or both. Cell death was measured by MTT and FACS. Western blot was used to measure autophagy by LC3 processing and apoptosis by caspase-3 cleavage. RRM2 expression was measured by western blot in cells treated with PEG-ADI. ASS expression was measured by western blot in cells treated with gemcitabine. To test the in vivo effect arginine deprivation a mice xenograft model was created by subcutaneously injecting Mia-PaCa2 cells into the flanks of 28 mice (two tumors per mice). Tumors were allowed to mature at which time the mice were assigned to one of four treatment groups: PBS control (once weekly), ADI-PEG20 (5 IU once weekly), Gemcitabine (125mg/kg twice weekly), or Gemcitabine and ADI-PEG20. All treatments were administered via intraperitoneal injection and tumors were measured twice a week. Results: 1. MIA-PaCa-2 lacks ASS and is auxotrophic for arginine. 2. PEG-ADI induces autophagy and cell death 3. Gemcitabine has little in vitro cytotoxicity 4. The combination of gemcitabine and PEG-ADI is at least additive in vitro and in vivo. 5. PEG-ADI does not regulate RRM2 expression and gemcitabine does not regulate ASS expression. Conclusions: Therapy targeting autophagy may provide increased efficacy for traditional genotoxic chemotherapies in pancreatic cancer. PEG-ADI in combination with gemcitabine is a novel combination to be pursued in clinical trials 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 4847.

Argininosuccinate Synthase Expression Is Required to Maintain Nitric Oxide Production and Cell Viability in Aortic Endothelial Cells

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.