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1.
Adv Sci (Weinh) ; 11(12): e2307256, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38233193

RESUMEN

Atherosclerosis is initiated with endothelial cell (EC) dysfunction and vascular inflammation under hyperlipidemia. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase. However, the specific role of endothelial SIRT3 during atherosclerosis remains poorly understood. The present study aims to study the role and mechanism of SIRT3 in EC function during atherosclerosis. Wild-type Sirt3f/f mice and endothelium-selective SIRT3 knockout Sirt3f/f; Cdh5Cre/+ (Sirt3EC-KO) mice are injected with adeno-associated virus (AAV) to overexpress PCSK9 and fed with high-cholesterol diet (HCD) for 12 weeks to induce atherosclerosis. Sirt3EC-KO mice exhibit increased atherosclerotic plaque formation, along with elevated macrophage infiltration, vascular inflammation, and reduced circulating L-arginine levels. In human ECs, SIRT3 inhibition resulted in heightened vascular inflammation, reduced nitric oxide (NO) production, increased reactive oxygen species (ROS), and diminished L-arginine levels. Silencing of SIRT3 results in hyperacetylation and deactivation of Argininosuccinate Synthase 1 (ASS1), a rate-limiting enzyme involved in L-arginine biosynthesis, and this effect is abolished in mutant ASS1. Furthermore, L-arginine supplementation attenuates enhanced plaque formation and vascular inflammation in Sirt3EC-KO mice. This study provides compelling evidence supporting the protective role of endothelial SIRT3 in atherosclerosis and also suggests a critical role of SIRT3-induced deacetylation of ASS1 by ECs for arginine synthesis.


Asunto(s)
Aterosclerosis , Sirtuina 3 , Humanos , Ratones , Animales , Proproteína Convertasa 9 , Argininosuccinato Sintasa , Arginina , Endotelio , Inflamación
2.
BMC Cancer ; 22(1): 864, 2022 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-35941558

RESUMEN

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.


Asunto(s)
Arginina , Neoplasias de la Mama , Leucil Aminopeptidasa/metabolismo , Animales , Arginina/metabolismo , Argininosuccinato Sintasa/metabolismo , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Bovinos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Interferón gamma/metabolismo
3.
Biochem Biophys Res Commun ; 611: 31-37, 2022 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-35477090

RESUMEN

Previous studies demonstrated that arginine biosynthesis was frequently impaired in acute liver injury. However, the underlying mechanisms remain elusive. In this study, we found that Argininosuccinate synthetase 1 (ASS1), a rate-limiting enzyme in arginine metabolism, was downregulated in the TAA-induced liver injury model. Single-cell RNA-seq data found that ASS1 was highly enriched in the hepatocytes. The reduction of ASS1 was attributed to the decreased expression of Farnesoid X receptor (FXR), which is a bile acid-activated nuclear hormone receptor with high expression in the liver. Subsequent studies demonstrated that activation of FXR by its agonist obeticholic acid (OCA) directly promoted ASS1 transcription and enhanced arginine synthesis, leading to the alleviation of TAA-mediated liver injury. Further experiments found that OCA, ASS1, and arginine supplement can rescue TAA-mediated hepatocytes apoptosis by decreasing the protein levels of Cyto C, PARP, and Caspase 3. Taken together, our study illustrated a protective role of the FXR/ASS1 axis in TAA-induced liver injury by targeting arginine metabolism, which might shed light on the development of novel therapeutic approaches for acute liver injury.


Asunto(s)
Arginina , Argininosuccinato Sintasa , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Receptores Citoplasmáticos y Nucleares , Animales , Arginina/metabolismo , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo
4.
Front Immunol ; 12: 653571, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34054815

RESUMEN

Macrophages are indispensable immune cells tasked at eliminating intracellular pathogens. Mycobacterium tuberculosis (Mtb), one of the most virulent intracellular bacterial pathogens known to man, infects and resides within macrophages. While macrophages can be provoked by extracellular stimuli to inhibit and kill Mtb bacilli, these host defense mechanisms can be blocked by limiting nutritional metabolites, such as amino acids. The amino acid L-arginine has been well described to enhance immune function, especially in the context of driving macrophage nitric oxide (NO) production in mice. In this study, we aimed to establish the necessity of L-arginine on anti-Mtb macrophage function independent of NO. Utilizing an in vitro system, we identified that macrophages relied on NO for only half of their L-arginine-mediated host defenses and this L-arginine-mediated defense in the absence of NO was associated with enhanced macrophage numbers and viability. Additionally, we observed macrophage glycolysis to be driven by both L-arginine and mechanistic target of rapamycin (mTOR), and inhibition of glycolysis or mTOR reduced macrophage control of Mtb as well as macrophage number and viability in the presence of L-arginine. Our data underscore L-arginine as an essential nutrient for macrophage function, not only by fueling anti-mycobacterial NO production, but also as a central regulator of macrophage metabolism and additional host defense mechanisms.


Asunto(s)
Arginina/metabolismo , Suplementos Dietéticos , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/dietoterapia , Animales , Arginina/administración & dosificación , Argininosuccinatoliasa/genética , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Cultivo Primario de Células , Células RAW 264.7 , Tuberculosis/inmunología , Tuberculosis/microbiología
5.
Anal Chem ; 92(17): 11505-11510, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32794704

RESUMEN

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.


Asunto(s)
Extractos Celulares/química , Citrulina/química , Ornitina/química , Proteínas/química , Secuencia de Aminoácidos , Arginina/química , Argininosuccinatoliasa/genética , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Transferasas de Carboxilo y Carbamoilo/genética , Transferasas de Carboxilo y Carbamoilo/metabolismo , Citrulina/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Ornitina/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica , Estereoisomerismo , Especificidad por Sustrato
6.
J Inherit Metab Dis ; 41(5): 777-784, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29651749

RESUMEN

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium-chain triglyceride (MCT) supplement therapy with a low-carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients' general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low-carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.


Asunto(s)
Carbohidratos/administración & dosificación , Citrulinemia/dietoterapia , Encefalopatía Hepática/dietoterapia , Hiperamonemia/dietoterapia , Triglicéridos/administración & dosificación , Anciano , Amoníaco/sangre , Amoníaco/metabolismo , Argininosuccinato Sintasa/metabolismo , Citrulinemia/complicaciones , Suplementos Dietéticos , Hígado Graso/etiología , Femenino , Alimentos Formulados , Hepatocitos/metabolismo , Humanos , Hiperamonemia/sangre , Trasplante de Hígado , Masculino , Persona de Mediana Edad
7.
Oncotarget ; 7(45): 73292-73308, 2016 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-27689335

RESUMEN

The moderate anticancer effect of arginine deprivation in clinical trials has been linked to an induced argininosuccinate synthetase (ASS1) expression in initially ASS1-negative tumors, and ASS1-positive cancers are anticipated as non-responders. Our previous studies indicated that arginine deprivation and low doses of the natural arginine analog canavanine can enhance radioresponse. However, the efficacy of the proposed combination in the presence of extracellular citrulline, the substrate for arginine synthesis by ASS1, remains to be elucidated, in particular for malignant cells with positive and/or inducible ASS1 as in colorectal cancer (CRC). Here, the physiological citrulline concentration of 0.05 mM was insufficient to overcome cell cycle arrest and radiosensitization triggered by arginine deficiency. Hyperphysiological citrulline (0.4 mM) did not entirely compensate for the absence of arginine and significantly decelerated cell cycling. Similar levels of canavanine-induced apoptosis were detected in the absence of arginine regardless of citrulline supplementation both in 2-D and advanced 3-D assays, while normal colon epithelial cells in organoid/colonosphere culture were unaffected. Notably, canavanine tremendously enhanced radiosensitivity of arginine-starved 3-D CRC spheroids even in the presence of hyperphysiological citrulline. We conclude that the novel combinatorial targeting strategy of metabolic-chemo-radiotherapy has great potential for the treatment of malignancies with inducible ASS1 expression.


Asunto(s)
Arginina/metabolismo , Canavanina/administración & dosificación , Citrulina/metabolismo , Radiación Ionizante , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de la radiación , Tolerancia a Radiación , Esferoides Celulares , Células Tumorales Cultivadas
8.
J Med Genet ; 53(10): 710-9, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27287393

RESUMEN

BACKGROUND: Citrullinemia type 1 is an autosomal-recessive urea cycle disorder caused by mutations in the ASS1 gene and characterised by increased plasma citrulline concentrations. Of the ∼90 argininosuccinate synthetase (ASS) missense mutations reported, 21 map near the substrate (aspartate or citrulline) binding site, and thus are potential kinetic mutations whose decreased activities could be amenable to substrate supplementation. This article aims at characterising these 21 ASS mutations to prove their disease-causing role and to test substrate supplementation as a novel therapeutic approach. METHODS: We used an Escherichia coli expression system to study all potentially kinetic ASS mutations. All mutant enzymes were nickel-affinity purified, their activity and kinetic parameters were measured using tandem mass spectrometry and their thermal stability using differential scanning fluorimetry. Structural rationalisation of the effects of these mutations was performed. RESULTS: Of the characterised mutants, 13 were totally inactive while 8 exhibited decreased affinity for aspartate and citrulline. The activity of these eight kinetic mutations could be rescued to ∼10-99% of the wild-type using high l-aspartate concentrations. CONCLUSIONS: Substrate supplementation raised in vitro the activity of eight citrullinemia type 1 mutations with reduced affinity for aspartate. As a direct translation of these results to the clinics, we propose to further evaluate the use of oxaloacetate, a nitrogen-free aspartate precursor and already available medical food (anti-ageing and brain stimulating, not considered as a drug by the US Food and Drug Administration), in patients with citrullinemia type 1 with decreased aspartate affinity. Although only patients with kinetic mutations would benefit, oxaloacetate could offer a safe novel treatment.


Asunto(s)
Argininosuccinato Sintasa/genética , Ácido Aspártico/uso terapéutico , Citrulinemia/enzimología , Argininosuccinato Sintasa/metabolismo , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Dominio Catalítico/genética , Citrulina/metabolismo , Citrulinemia/tratamiento farmacológico , Citrulinemia/genética , Humanos , Cinética , Mutación Missense
9.
Cancer Biol Ther ; 16(7): 1047-55, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25774632

RESUMEN

Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50%). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70% killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Arginina/metabolismo , Glioblastoma/tratamiento farmacológico , Hidrolasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Argininosuccinatoliasa/genética , Argininosuccinato Sintasa/genética , Benzopiranos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cloroquina/administración & dosificación , Metilación de ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Hidrolasas/administración & dosificación , Hidrolasas/metabolismo , Ácidos Hidroxámicos/administración & dosificación , Masculino , Ratones Endogámicos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptococcus pyogenes/enzimología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Vorinostat
10.
PLoS One ; 10(3): e0121224, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25822522

RESUMEN

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.


Asunto(s)
Nivel de Alerta/fisiología , Metabolismo Energético/fisiología , Estivación/fisiología , Peces/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Hígado/metabolismo , Redes y Vías Metabólicas/fisiología , África , Animales , Argininosuccinato Sintasa/metabolismo , Secuencia de Bases , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , ADN Complementario/genética , Peces/fisiología , Biblioteca de Genes , Datos de Secuencia Molecular , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Superóxido Dismutasa/metabolismo
11.
J Comp Physiol B ; 184(7): 835-53, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25034132

RESUMEN

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.


Asunto(s)
Argininosuccinatoliasa/genética , Argininosuccinato Sintasa/genética , Estivación/genética , Peces/genética , Secuencia de Aminoácidos , Animales , Arginina/sangre , Arginina/metabolismo , Argininosuccinatoliasa/fisiología , Argininosuccinato Sintasa/fisiología , Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario/genética , Estivación/fisiología , Peces/fisiología , Riñón/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , Filogenia , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN
12.
Mol Genet Metab ; 107(3): 247-52, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22819233

RESUMEN

Mitochondrial diseases arise as a result of dysfunction of the respiratory chain, leading to inadequate ATP production required to meet the energy needs of various organs. On the other hand, nitric oxide (NO) deficiency can occur in mitochondrial diseases and potentially play major roles in the pathogenesis of several complications including stroke-like episodes, myopathy, diabetes, and lactic acidosis. NO deficiency in mitochondrial disorders can result from multiple factors including decreased NO production due to endothelial dysfunction, NO sequestration by cytochrome c oxidase, NO shunting into reactive nitrogen species formation, and decreased availability of the NO precursors arginine and citrulline. Arginine and citrulline supplementation can result in increased NO production and hence potentially have therapeutic effects on NO deficiency-related manifestations of mitochondrial diseases. Citrulline is a more efficient NO donor than arginine as it results in a greater increase in de novo arginine synthesis, which plays a major role in driving NO production. This concept is supported by the observation that the three enzymes responsible for recycling citrulline to NO (argininosuccinate synthase and lyase, and nitric oxide synthase) function as a complex that can result in compartmentalizing NO synthesis and channeling citrulline efficiently to NO synthesis. Clinical research evaluating the effect of arginine and citrulline in mitochondrial diseases is limited to uncontrolled open label studies demonstrating that arginine administration to subjects with MELAS syndrome results in improvement in the clinical symptoms associated with stroke-like episodes and a decrease in the frequency and severity of these episodes. Therefore, controlled clinical studies of the effects of arginine or citrulline supplementation on different aspects of mitochondrial diseases are needed to explore the potential therapeutic effects of these NO donors.


Asunto(s)
Arginina/uso terapéutico , Citrulina/uso terapéutico , Síndrome MELAS/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Óxido Nítrico/deficiencia , Arginina/metabolismo , Arginina/farmacología , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Citrulina/metabolismo , Citrulina/farmacología , Ensayos Clínicos como Asunto , Transporte de Electrón/efectos de los fármacos , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Óxido Nítrico/agonistas , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especies de Nitrógeno Reactivo/metabolismo
13.
Anticancer Drugs ; 23(1): 51-64, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21955999

RESUMEN

Human recombinant arginase I cobalt [HuArgI (Co)] coupled with polyethylene glycol 5000 [HuArgI (Co)-PEG5000] has shown potent in-vitro depletion of arginine from tissue culture medium. We now show that HuArgI (Co)-PEG5000 is toxic to almost all cancer cell lines and to some normal primary cells examined. In contrast, HuArgI (Co)-PEG5000 in combination with supplemental L-citrulline is selectively cytotoxic to a fraction of human cancer cell lines in tissue culture, including some melanomas, mesotheliomas, acute myeloid leukemias, hepatocellular carcinomas, pancreas adenocarcinomas, prostate adenocarcinomas, lung adenocarcinomas, osteosarcomas, and small cell lung carcinomas. Unfortunately, a subset of normal human tissues is also sensitive to HuArgI (Co)-PEG5000 with L-citrulline supplementation, including umbilical endothelial cells, bronchial epithelium, neurons, and renal epithelial cells. We further show that cell sensitivity is predicted by the level of cellular argininosuccinate synthetase protein expression measured by immunoblots. By comparing a 3-day and 7-day exposure to HuArgI (Co)-PEG5000 with supplemental L-citrulline, some tumor cells sensitive on short-term assay are resistant in the 7-day assay consistent with the induction of argininosuccinate synthetase expression. On the basis of these results, we hypothesize that HuArgI (Co)-PEG5000 in combination with L-citrulline supplementation may be an attractive therapeutic agent for some argininosuccinate synthetase-deficient tumors. These in-vitro findings stimulate further development of this molecule and may aid in the identification of tissue toxicities and better selection of patients who will potentially respond to this combination therapy.


Asunto(s)
Antineoplásicos/farmacología , Arginasa/farmacología , Argininosuccinato Sintasa/metabolismo , Citrulina/farmacología , Polietilenglicoles/farmacología , Arginina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Medios de Cultivo , Ensayos de Selección de Medicamentos Antitumorales , Células Epiteliales/efectos de los fármacos , Humanos , Masculino , Ornitina Carbamoiltransferasa/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/farmacología
14.
Mol Plant Pathol ; 11(5): 613-24, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20696000

RESUMEN

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the beta-tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446-bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of L-arginine, inadvertent gene silencing of bcass1 may result in partial L-arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild-type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or beta-glucuronidase using the pLOB1-based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.


Asunto(s)
Argininosuccinato Sintasa/genética , Botrytis/enzimología , Botrytis/genética , Silenciador del Gen , Genes Fúngicos/genética , Vectores Genéticos/genética , Arginina/farmacología , Argininosuccinato Sintasa/metabolismo , Bioensayo , Botrytis/efectos de los fármacos , Botrytis/patogenicidad , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Prueba de Complementación Genética , Sitios Genéticos/genética , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/microbiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/efectos de los fármacos , Reproducibilidad de los Resultados , Transformación Genética/efectos de los fármacos , Virulencia/efectos de los fármacos
15.
Surgery ; 148(2): 310-8, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20466402

RESUMEN

BACKGROUND: Hepatocellular and pancreatic carcinomas are often auxotrophic for L-arginine, a semi-essential amino acid. The purpose of this study was to investigate cancer cell death using a significantly more active, cobalt-substituted bioengineered arginase. METHODS: Panc-1, a human pancreatic carcinoma cell line, and Hep 3B, a human hepatocellular carcinoma cell line, were exposed to L-arginase. Flow cytometry was used to measure expression of Ki-67, caspase-3, and argininosuccinate synthetase-1 (ASS-1) 4 days after treatment. An MTT assay measured proliferation. The Student t test determined statistical significance. RESULTS: Viability decreased by 31% +/- 2% for Panc-1 cells (P < .0001) and 34% +/- 1% (P < .0001) for Hep 3B cells after treatment. Both cell lines demonstrated a 4-fold increase activated caspase-3 expression after high dose treatment (P < .01), and 5-fold increase in ASS-1 expression (P < .002). Ki-67 expression did not vary in Hep 3B cells but decreased for Panc-1 cells (P < .015). The 50% inhibitory concentration was 8-fold higher for Panc-1 cells than for Hep 3B cells (P < .03). CONCLUSION: Increased ASS-1 expression by these cells, in order to increase L-arginine concentration, is inadequate, suggesting a mechanism by which arginine depletion can be used in multimodality therapy for arginine-dependent cancers.


Asunto(s)
Arginasa/farmacología , Argininosuccinato Sintasa/biosíntesis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/enzimología , Caspasa 3/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/enzimología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Arginasa/administración & dosificación , Arginina/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/patología , Modelos Biológicos , Neoplasias Pancreáticas/patología , Ingeniería de Proteínas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología
16.
Hypertension ; 50(6): 1077-84, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17938381

RESUMEN

NO deficiency is associated with development of hypertension. Defects in the renal citrulline-arginine pathway or arginine reabsorption potentially reduce renal NO in prehypertensive spontaneously hypertensive rats (SHRs). Hence, we investigated genes related to the citrulline-arginine pathway or arginine reabsorption, amino acid pools, and renal NO in 2-week-old prehypertensive SHRs. In addition, because perinatally supporting NO availability reduces blood pressure in SHRs, we supplemented SHR dams during pregnancy and lactation with citrulline, the rate-limiting amino acid for arginine synthesis. In female offspring, gene expression of argininosuccinate synthase (involved in renal arginine synthesis) and renal cationic amino acid Y-transporter (involved in arginine reabsorption) were both decreased in 2-day and 2-week SHRs compared with normotensive WKY, although no abnormalities in amino acid pools were observed. In addition, 2-week-old female SHRs had much less NO in their kidneys (0.46+/-0.01 versus 0.68+/-0.05 nmol/g of kidney weight, respectively; P<0.001) but not in their heart. Furthermore, perinatal supplementation with citrulline increased renal NO to 0.59+/-0.02 nmol/g of kidney weight (P<0.001) at 2 weeks and persistently ameliorated the development of hypertension in females and until 20 weeks in male SHR offspring. Defects in both the renal citrulline-arginine pathway and in arginine reabsorption precede hypertension in SHRs. We propose that the reduced cationic amino acid transporter disables the developing SHR kidney to use arginine reabsorption to compensate for reduced arginine synthesis, resulting in organ-specific NO deficiency. This early renal deficiency and its adverse sequels can be corrected by perinatal citrulline supplementation persistently in female and transiently in male SHRs.


Asunto(s)
Antihipertensivos/administración & dosificación , Citrulina/administración & dosificación , Riñón/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Óxido Nítrico/biosíntesis , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/análisis , Animales , Arginina/metabolismo , Argininosuccinato Sintasa/genética , Presión Sanguínea/efectos de los fármacos , Citrulina/metabolismo , Femenino , Perfilación de la Expresión Génica , Riñón/química , Masculino , Óxido Nítrico/análisis , Óxido Nítrico/deficiencia , Óxido Nítrico Sintasa/genética , Embarazo , Ratas , Ratas Endogámicas SHR
17.
Exp Neurol ; 208(1): 100-9, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17900569

RESUMEN

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.


Asunto(s)
Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Encéfalo/enzimología , Ataque Isquémico Transitorio/enzimología , Microglía/enzimología , Animales , Argininosuccinatoliasa/análisis , Argininosuccinato Sintasa/análisis , Infarto Cerebral/etiología , Infarto Cerebral/patología , Cuerpo Estriado/química , Cuerpo Estriado/patología , Lóbulo Frontal/química , Lóbulo Frontal/patología , Ataque Isquémico Transitorio/complicaciones , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Masculino , Microglía/química , Microglía/patología , Examen Neurológico , Neuronas/química , Neuronas/patología , Óxido Nítrico Sintasa de Tipo I/análisis , Lóbulo Parietal/química , Lóbulo Parietal/patología , Ratas , Ratas Wistar
18.
J Biochem Mol Biol ; 39(4): 400-5, 2006 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-16889683

RESUMEN

A 16-month old boy was referred to our hospital for evaluation of recurrent generalized tonic clonic seizures. Metabolic evaluation revealed significant hyperammonemia (1,112 microg/dl). Amino acid/acylcarnitine screening using tandem mass spectrometry showed markedly increased plasma levels of citrulline (1,350 microM/l) with undetectable levels of arginine and arginosuccinic acid. Urinary excretion of citrulline was markedly increased (38,617 microM/g creatinine). Brain MRI findings showed diffuse high-signal intensity lesions, that involved gray and white matter in both frontal lobes and insula with edematous changes; these findings were consistent with the acute stage of citrullinemia (CTLN). Mutation analysis of the argininosuccinate synthetase (ASS) gene, in this patient, showed a Gly324Ser mutation in exon 13, and a 67-bp duplication mutation in exon 15 (c.1128-6_1188dup67). The patient was confirmed as having late-onset CTLN1 and treated with anticonvulsants, lactulose enema, protein restricted diet and arginine. Here we describe a case of late-onset CTLN1 in a patient by biochemical analyses and ASS gene mutation confirmation. This is the first report of a Korean patient with late-onset CTLN1 confirmed by ASS gene mutation identification.


Asunto(s)
Argininosuccinato Sintasa/genética , Citrulinemia/genética , Encéfalo/patología , Citrulina/sangre , Citrulina/orina , Citrulinemia/metabolismo , Citrulinemia/patología , Análisis Mutacional de ADN , Exones , Femenino , Duplicación de Gen , Humanos , Lactante , Corea (Geográfico) , Masculino , Mutación Missense , Convulsiones/genética , Convulsiones/metabolismo
19.
Mol Genet Metab ; 89(3): 274-6, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16580861

RESUMEN

Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was increased in all patients, while cerebral creatine was low in ASS and high in ASL deficiency. Because high cerebral guanidinoacetate might be toxic, lowering the arginine supplementation with additional creatine supplementation might be important.


Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Argininosuccinato Sintasa/deficiencia , Aciduria Argininosuccínica , Encéfalo/metabolismo , Creatina/metabolismo , Glicina/análogos & derivados , Arginina/sangre , Niño , Preescolar , Creatina/sangre , Creatina/orina , Femenino , Glicina/sangre , Glicina/metabolismo , Glicina/orina , Humanos , Recién Nacido , Masculino , Embarazo
20.
J Hypertens ; 22(8): 1487-94, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15257170

RESUMEN

OBJECTIVE: Inherited differences in renal function underlie the effect of high salt diets on blood pressure in Dahl rats. We probed the kidneys of inbred Dahl SS/Jr and SR/Jr for anonymous and candidate genes whose expression was regulated by dietary sodium. METHODS: mRNA quantitation of both candidate genes implicated in sodium excretion and anonymous gene products found by differential hybridization in the kidneys of salt-resistant (SR) and salt sensitive (SS) inbred Dahl rats on high and low salt diets for 21 days. RESULTS: Differential screening revealed a cDNA clone (H1) that showed increased dietary salt-dependent expression only in SS rats. Sequencing of the H1 cDNA showed it was the Dahl rat homologue to a perchloric acid soluble protein expressed in liver and kidney. Among candidate genes, transcript levels of arginosuccinate synthetase (AS) and arginosuccinate lyase (AL) were higher in SS on low salt diets, and AS mRNA increased in response to a high salt diet in SR. Renal mRNA for the ANP-A and the vasopressin type II receptors did not differ by strain or dietary conditions. CONCLUSIONS: Three new salt-sensitive genes were detected in the kidneys of inbred Dahl rats. Two genes encode enzymes in the biosynthesis of L-arginine. The upregulation of these genes by dietary salt indicates increased demand and biosynthesis of L-arginine in Dahl SS rats. A third gene encodes a small acid-soluble protein thought to influence the transcription/translation of numerous genes. Further studies will be needed to determine the nature of the association of these genes with salt-sensitivity and blood pressure.


Asunto(s)
Pruebas Genéticas , Hipertensión/genética , Riñón/fisiología , Ratas Endogámicas Dahl/genética , Animales , Arginina/biosíntesis , Argininosuccinatoliasa/genética , Argininosuccinato Sintasa/genética , Presión Sanguínea/genética , Northern Blotting , ADN Complementario , Hibridación Genética , Masculino , Ratas , Cloruro de Sodio Dietético/farmacología
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