RESUMEN
CONTEXT: Geometrical knots are rare structural arrangements in proteins in which the polypeptide chain ties itself into a knot, which is very intriguing due to the uncertainty of their impact on the protein properties. Presently, classical molecular dynamics is the most employed technique in the few studies found on this topic, so any information on how the presence of knots affects the reactivity and electronic properties of proteins is even scarcer. Using the electronic structure methods and quantum chemical descriptors analysis, we found that the same amino-acid residues in the knot core have statistically larger values for the unknotted protein, for both hard-hard and soft-soft interaction descriptors. In addition, we present a computationally feasible protocol, where we show it is possible to separate the contribution of the geometrical knot to the reactivity and other electronic structure properties. METHODS: In order to investigate these systems, we used PRIMoRDiA, a new software developed by our research group, to explore the electronic structure of biological macromolecules. We evaluated several local quantum chemical descriptors to unveil relevant patterns potentially originating from the presence of the geometrical knot in two proteins, belonging to the ornithine transcarbamylase family. We compared several sampled structures from these two enzymes that are highly similar in both tertiary structure and function, but one of them has a knot whereas the other does not. The sampling was carried out through molecular dynamics simulations using ff14SB force field along 50 ns, and the semiempirical convergence was performed with PM7 Hamiltonian.
Asunto(s)
Simulación de Dinámica Molecular , Ornitina Carbamoiltransferasa , Ornitina Carbamoiltransferasa/química , Ornitina Carbamoiltransferasa/metabolismo , Conformación Proteica , Modelos MolecularesRESUMEN
PFstats is a software developed for the extraction of useful information from protein multiple sequence alignments. By analyzing positional conservation and residue coevolution networks, the software allows the identification of structurally and functionally important residue groups and the discovery of probable functional subclasses. Furthermore, it contains tools for the identification of the possible biological significance of these findings. PFstats contains methods for maximizing the significance of alignments through filtering and weighting, residue conservation and coevolution analysis, automatic UniprotKb queries for residue-position annotation and many possible data visualization methods.
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Aspartato Carbamoiltransferasa/metabolismo , Citrato (si)-Sintasa/metabolismo , Familia de Multigenes , Ornitina Carbamoiltransferasa/metabolismo , Mapas de Interacción de Proteínas , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Aspartato Carbamoiltransferasa/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Citrato (si)-Sintasa/química , Biología Computacional , Bases de Datos de Proteínas , Humanos , Ornitina Carbamoiltransferasa/químicaRESUMEN
INTRODUCTION: This work focuses on ammonia metabolism of Liver Microorgans (LMOs) after cold preservation in a normothermic reoxygenation system (NRS). We have previously reported the development of a novel preservation solution, Bes-Gluconate-PEG 35 kDa (BG35) that showed the same efficacy as ViaSpan to protect LMOs against cold preservation injury. The objective of this work was to study mRNA levels and activities of two key Urea Cycle enzymes, Carbamyl Phosphate Synthetase I (CPSI) and Ornithine Transcarbamylase (OTC), after preservation of LMOs in BG35 and ViaSpan and the ability of these tissue slices to detoxify an ammonia overload in a NRS model. MATERIAL AND METHODS: After 48 h of cold storage (0°C in BG35 or ViaSpan) LMOs were rewarmed in KHR containing an ammonium chloride overload (1 mM). We determined ammonium detoxification capacity (ADC), urea synthesis and enzyme activities and relative mRNA levels for CPSI and OTC. RESULTS: At the end of reoxygenation LMOs cold preserved in BG35 have ADC and urea synthesis similar to controls. ViaSpan group demonstrated a lower capacity to detoxify ammonia and to synthesize urea than fresh LMOs during the whole reoxygenation period which correlated with the lower mRNA levels and activities for CPSI and OTC observed for this group. CONCLUSION: We demonstrate that our preservation conditions (48 hours, BG35 solution, anoxia, 0ºC) did not affect ammonia metabolism of cold preserved LMOs maintaining the physiological and biochemical liver functions tested, which allows their future use as biological component of a BAL system.
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Amoníaco/metabolismo , Frío , Hígado/efectos de los fármacos , Hígado/metabolismo , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Adenosina/farmacología , Alopurinol/farmacología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Glutatión/farmacología , Insulina/farmacología , Hígado/patología , Pruebas de Función Hepática , Masculino , Modelos Animales , Ornitina Carbamoiltransferasa/metabolismo , Oxígeno/administración & dosificación , Oxígeno/metabolismo , ARN Mensajero/metabolismo , Rafinosa/farmacología , Ratas , Ratas Wistar , Reperfusión , Factores de TiempoRESUMEN
The present study was aimed to assess the effect of protein carbonylation (PC) in hepatic cells and effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on indicators of tissue damage induced by liver ischemia-reperfusion injury (LIRI). Warm ischemia was performed by partial vascular occlusion during 90 min in Wistar rats. In serum, we determined the catalytic activity of Alanine Aminotransferase, Aspartate Aminotransferase, Lacticate Dehydrogenase, and Ornithine Carbamoyltransferase. In liver samples, we studied cellular alterations by means of histologic studies, lipid peroxidation, PC by immunohistochemistry, apoptosis and reactive oxygen species in bile by electron paramagnetic resonance. Based on PC data, sinusoidal endothelial cells (SEC) and Kupffer cells (KC) were the first to exhibit LIRI-associated oxidative damage and prior to parenchymal cells. Administration of piroxicam or meloxicam during the pre-ischemic period produced a highly significant decrease in all studied injury indicators. No significant differences were revealed between the protective action of the two drugs. The data shown here suggest the potential use of NSAIDs such as piroxicam or meloxicam in minimizing ischemic event-caused damage in liver. We also propose that PC may be employed as an adequate tool to assess tissue damage after oxidative stress.
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Carbono/química , Células Endoteliales/citología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Piroxicam/farmacología , Daño por Reperfusión , Tiazinas/farmacología , Tiazoles/farmacología , Alanina Transaminasa/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspartato Aminotransferasas/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Meloxicam , Ornitina Carbamoiltransferasa/metabolismo , Estrés Oxidativo , Proteínas/metabolismo , Ratas , Ratas WistarRESUMEN
UNLABELLED: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups. IN CONCLUSION: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.
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Butileno Glicoles/farmacología , Criopreservación/métodos , Crioprotectores/farmacología , Hepatocitos/efectos de los fármacos , Soluciones Preservantes de Órganos/farmacología , Urea/metabolismo , Adenosina/farmacología , Alopurinol/farmacología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/farmacología , Hepatocitos/enzimología , Insulina/farmacología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Propidio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , ARN Mensajero/metabolismo , Rafinosa/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The present work reports the activities of urea cycle enzymes during the ontogenic development of the teleost pacu (Piaractus mesopotamicus). Urea cycle enzymes from the kidney and liver of adult fish were compared with those from the fish's embryonic phases. Samples were evaluated over all phases of embryonic development, the larval period and alevin. Ammonia and urea concentrations were determined during embryogenesis and in the plasma of adult fish. Except for carbamoyl phosphate synthetase-III (CPS-III), all enzymes of the urea cycle were expressed in the larvae and alevins as well as in the liver and kidney of adult fish. In spite of the low level of activity of the ornithine urea cycle (OUC) enzymes compared to those in mammals, and the low levels of tissue urea concentration compared to ammonia, the ureogenesis was evaluated in pacu. Ammonia seems to be the main nitrogenous waste during embryonic development. In this phase glutamine synthetase (GS) may play a role in ammonia detoxification, and the OUC enzymes can be individually involved in functions other than urea production. The presence of ornithine carbamoyl transferase (OCT) in all developmental phases of pacu and in the adult liver and kidney suggests that this enzyme is performing different metabolic pathways. OCT in the kidney, wherein the activity is less than in the liver, should work in the biosynthesis of polyamines and control the arginine plasma concentration given that renal arginase and argininosuccinate synthetase-argininosuccinate lyase are more active than from the liver. We suppose that OCT during the embryogenesis is a control step regulating the cellular concentration of ornithine for polyamines synthesis.
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Embrión no Mamífero/enzimología , Peces/crecimiento & desarrollo , Peces/metabolismo , Ornitina Carbamoiltransferasa/metabolismo , Urea/metabolismo , Amoníaco/sangre , Animales , Análisis Químico de la Sangre , Femenino , Peces/embriología , Concentración de Iones de Hidrógeno , Riñón/enzimología , Cinética , Larva/enzimología , Hígado/enzimología , Masculino , Temperatura , Urea/sangreRESUMEN
Urea cycle (UC) is the main pathway of ammonium removal. A deficiency in any of the five classical enzymes of the pathway causes a urea cycle disorder. Hepatocellular transplantation is one of the techniques applicable to treat this disorder. In the present work, we investigated the activities and the relative expression levels of two of the UC enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), in isolated hepatocytes preserved up to 120 h in University of Wisconsin (UW) solution at 0 degrees C, and during the rewarming of these suspensions. During preservation, CPSI showed differences in mRNA levels respect to time 0, while ornithine transcarbamylase remained unchanged. At the end of the rewarming, CPSI showed values of enzymatic activity and relative mRNA level comparable with the control, meanwhile, there was an increment in OTC activity. In line with these results, we found that hepatocytes cold preserved up to 120h in UW solution maintained their ability to remove an ammonium load comparable to freshly isolated hepatocytes. These data indicated that our preservation conditions up to 120h in UW solution followed by rewarming, preserves UC enzymes at levels similar to freshly isolated hepatocytes, allowing the use of these cells in bioartificial liver devices or hepatocellular transplantation.
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Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Frío , Hepatocitos/metabolismo , Ornitina Carbamoiltransferasa/metabolismo , Conservación de Tejido , Adenosina , Alopurinol , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Células Cultivadas , Expresión Génica , Glutatión , Hepatocitos/enzimología , Insulina , L-Lactato Deshidrogenasa/metabolismo , Masculino , Soluciones Preservantes de Órganos , Ornitina Carbamoiltransferasa/genética , Compuestos de Amonio Cuaternario/metabolismo , ARN Mensajero/análisis , Rafinosa , Ratas , Ratas Wistar , Urea/metabolismoAsunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Criopreservación , Hepatocitos/enzimología , Soluciones Preservantes de Órganos/farmacología , Ornitina Carbamoiltransferasa/genética , Urea/metabolismo , Adenosina/farmacología , Alopurinol/farmacología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Regulación Enzimológica de la Expresión Génica , Glutatión/farmacología , Hepatocitos/citología , Insulina/farmacología , Masculino , Ornitina Carbamoiltransferasa/metabolismo , Rafinosa/farmacología , Ratas , Ratas WistarRESUMEN
Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.
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Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Ornitina Carbamoiltransferasa/metabolismo , Precursores de Proteínas/metabolismo , Temperatura , Proteínas Bacterianas/metabolismo , Carbamoil Fosfato/metabolismo , Medios de Cultivo , Exotoxinas/biosíntesis , Exotoxinas/farmacología , Mutación , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/genética , Pseudomonas syringae/efectos de los fármacos , Pseudomonas syringae/genética , Pseudomonas syringae/crecimiento & desarrollo , Pseudomonas syringae/metabolismoRESUMEN
Arginine deiminase activity increased in the presence of arginine in Lactobacillus plantarum strains N4 and N8 isolated from orange. The influence of citrulline and ornithine on arginine deiminase and ornithine transcarbamylase activities was strain-dependent. The growth and arginine and citrulline metabolism of L. plantarum were studied in the presence of tomato juice. Its addition enhances the growth in both strains. The specific amino acids utilization was inversely proportional to the initial glucose concentration. Arginine and citrulline addition to basal medium exerted a stimulatory effect on the growth of N4 strain, and this effect was observed only with citrulline in strain N8. The magnitude of this effect was lower in the presence of tomato juice.
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Arginina/metabolismo , Citrulina/metabolismo , Lactobacillus/crecimiento & desarrollo , Lactobacillus/metabolismo , Secuencia de Bases , Bebidas , Citrus/microbiología , Medios de Cultivo , ADN Ribosómico/química , Hidrolasas/metabolismo , Lactobacillus/enzimología , Lactobacillus/genética , Solanum lycopersicum , Datos de Secuencia Molecular , Ornitina Carbamoiltransferasa/metabolismo , ARN Ribosómico 16S/genéticaRESUMEN
Inborn errors of urea synthesis can present in the newborn period as a catastrophic illness or later in childhood or adulthood with an indolent course punctuated by hyperammonemic episodes. Because symptoms mimic other neuropsychiatric disorders, it is common for there to be a delay in diagnosis, often with dire consequences. Diagnosis relies on the combination of clinical suspicion and the measurement of ammonium, lactate, and amino acids in plasma and organic acids and orotic acid in urine. Treatment involves nitrogen restriction combined with the stimulation of alternate pathways of waste nitrogen excretion. More recently liver transplantation has been performed as enzyme replacement therapy. The outcome is poor in children who survive prolonged neonatal hyperammonemic coma, with most manifesting developmental disabilities. The etiology of neuronal injury in this disorder is unclear but may involve some combination of ammonia/amino acid accumulation, neurotransmitter alterations, and excitotoxic injury. Gene therapy holds the promise of improved treatment in the future.
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Errores Innatos del Metabolismo/metabolismo , Urea/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Amoníaco/sangre , Amoníaco/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Diagnóstico Diferencial , Humanos , Recién Nacido , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/terapia , Neurotransmisores/metabolismo , Ornitina Carbamoiltransferasa/metabolismoRESUMEN
The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) from Pseudomonas syringae pv. phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated Mr of 36,520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by the P. aeruginosa argF and the Escherichia coli argI and argF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be named argK.
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Exotoxinas/farmacología , Genes Bacterianos , Ornitina Carbamoiltransferasa/genética , Pseudomonas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Ornitina/análogos & derivados , Ornitina Carbamoiltransferasa/metabolismo , Regiones Promotoras Genéticas , Pseudomonas/efectos de los fármacos , Pseudomonas/enzimología , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transformación BacterianaRESUMEN
The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.
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Escherichia coli/fisiología , Ornitina/biosíntesis , Poliaminas/fisiología , Aminoácidos/fisiología , Escherichia coli/crecimiento & desarrollo , Ornitina/toxicidad , Ornitina Carbamoiltransferasa/metabolismo , Ornitina-Oxo-Ácido Transaminasa/metabolismoRESUMEN
Enzyme activities and DNA content have been measure in axolotl liver during the metamorphic period (4-8 months after spawning). Three different types of enzyme activity profiles were observed. In the type I profile (carbamoyl-phosphate synthase, arginase, ornithine transcarbamoylase, and glutamate dehydrogenase) enzyme activity is high in the youngest animals studied, and shows a minimum at 5 months followed by a maximum at 8 months of age. Thereafter activities do not change or slightly decrease. In the type II profile (tyrosine aminotransferase, glucose-6-phosphatase) enzyme activity shows a peak at 5 months of age and is low thereafter. Hexokinase, the enzyme with a type III profile, shows high activity throughout the metamorphic period. DNA content remains high throughout the metamorphic period but decreases 50% between 9 and 12 months of age, probably due to an increase in the size of the hepatocytes. No glucokinase activity was detected. High activities of cluster II enzymes represent early metamorphic events, while the rising part of cluster I is associated with late metamorphic events. The apparent molecular specific activity increases during natural development between 5 and 9 months of age, or precociously, upon thyroid hormone treatment. This change in apparent molecular specific activity is correlated to the advent of ureotelism.
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Ambystoma mexicanum/fisiología , Ambystoma/fisiología , Hígado/enzimología , Metamorfosis Biológica , Triyodotironina/fisiología , Envejecimiento , Animales , Arginasa/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , ADN/metabolismo , Femenino , Glucosa-6-Fosfatasa/metabolismo , Glutamato Deshidrogenasa/metabolismo , Hexoquinasa/metabolismo , Hígado/metabolismo , Masculino , Ornitina Carbamoiltransferasa/metabolismo , Tirosina Transaminasa/metabolismoRESUMEN
Activities of five urea cycle enzymes, carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, were measured in liver of rats fed two types of diet: diet I: raw common corn and, diet II: made of a mixture of corn starch ("maicena"), "chuño (dried potato), sugar and fat. The activities obtained were compared with those of a group of control rats fed a balanced diet with an adequate protein content. Diets were administered after weaning, through an experimental period of 90 days, and enzymatic activities were measured at regular intervals during this time. Animals raised on diet I as well as those raised on diet II, showed low activity of the five urea cycle enzymes, but the decrease of enzymatic activity was more pronounced and appeared earlier in animals raised on diet II. Carbamyl phosphate synthetase and argininosuccinate synthetase, the two synthetases of this cycle, decreased earlier and in a greater degree than the other three enzymes in animals raised on these two types of diet.