RESUMEN
PURPOSE: To identify the causes of prolonged length of hospital stay (LOHS) of patients treated with flexible ureteroscopy (fURS). The secondary endpoint was to identify the predictors of complications. METHODS: A retrospective single-center cohort study was conducted between January 2011 and December 2015. All consecutive patients treated with a planned fURS, regardless of the indication, AND stayed hospitalized for at least one additional day compared to the traditional postoperative LOHS of our center (=1 day) were included. A multivariate analysis was performed to investigate predictive factors of prolonged LOHS. RESULTS: Two hundred and seventy-two patients were included in the final analysis. The median duration of LOHS was two days [2-18]. Forty-seven percent of patients were discharged beyond day 1 and had no complication. Among them, 56% stayed for simple surveillance decided by the surgeon without any specific treatment introduced and 52% stayed for continuation of intravenous preoperative antibiotics. Of the remaining 144 patients who had a complication, 85.4% (123/144) had a minor complication (Clavien 1 or 2). In multivariate analysis, predictive factors of prolonged LOHS (discharge after day 1) were neurological comorbidities (paraplegia, spina bifida, multiple sclerosis) with an odds ratio of 4.39 [1.7; 11.4]. CONCLUSIONS: The causes of prolonged LOHS were mainly related to comorbidities. A number of patients stayed hospitalized without complications. The identification of predictive factors of complications and prolonged LOHS may allow better selection of patients eligible for outpatient surgery and select those for whom inpatient surgery is still recommended. LEVEL OF EVIDENCE: 3.
Asunto(s)
Hospitalización/estadística & datos numéricos , Tiempo de Internación/estadística & datos numéricos , Complicaciones Posoperatorias/epidemiología , Ureteroscopía/estadística & datos numéricos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Selección de Paciente , Estudios Retrospectivos , Ureteroscopía/efectos adversos , Adulto JovenRESUMEN
The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3kcal/mol to set the enzyme into a catalytically competent form.
Asunto(s)
Ácido Glutámico/química , Lisina/química , Fosfogluconato Deshidrogenasa/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Dominio Catalítico , Ácido Glutámico/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Fosfogluconato Deshidrogenasa/metabolismo , Unión Proteica , Protones , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Termodinámica , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔCp, with NADP binding ΔCp closer to what is expected of crystallographic structures, while NADPH ΔCp is three times larger. The estimated dimer-tetramer association constant is 1.5·10(6)M(-1), and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.
Asunto(s)
NADP/química , Fosfogluconato Deshidrogenasa/química , Multimerización de Proteína/fisiología , Trypanosoma brucei brucei/enzimología , Animales , NADP/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Ovinos , Especificidad de la Especie , Tripanosomiasis Africana/enzimologíaRESUMEN
A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.
Asunto(s)
Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Simulación por Computador , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Modelos Moleculares , Fosfogluconato Deshidrogenasa/química , Programas InformáticosRESUMEN
Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.
Asunto(s)
Proteínas de Unión al GTP/química , Transglutaminasas/química , Calorimetría , Dicroismo Circular , Eritrocitos , Calor , Humanos , Concentración de Iones de Hidrógeno , Cinética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteína Glutamina Gamma Glutamiltransferasa 2 , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Espectrometría de Fluorescencia , Temperatura , Factores de TiempoRESUMEN
A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1. 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.
Asunto(s)
Arginina/fisiología , Lactococcus lactis/enzimología , NADP/metabolismo , NAD/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/genética , Bacillus subtilis/enzimología , Sitios de Unión/genética , Escherichia coli/genética , Genes Bacterianos , Prueba de Complementación Genética , Isoenzimas/metabolismo , Cinética , Lactococcus lactis/genética , Datos de Secuencia Molecular , Fosfogluconato Deshidrogenasa/genética , Ovinos , Especificidad por Sustrato/genética , Trypanosoma brucei brucei/enzimologíaRESUMEN
Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Eritrocitos/enzimología , GTP Fosfohidrolasas/antagonistas & inhibidores , Proteínas de Unión al GTP , Transglutaminasas/antagonistas & inhibidores , o-Ftalaldehído/metabolismo , Sitios de Unión , Humanos , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.
Asunto(s)
Fosfogluconato Deshidrogenasa/metabolismo , Regulación Alostérica , Animales , Eritrocitos/enzimología , Escherichia coli/enzimología , Gluconatos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Hígado/enzimología , NADP/fisiología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Ovinos , Trypanosoma brucei brucei/enzimologíaRESUMEN
o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.
Asunto(s)
Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/metabolismo , o-Ftalaldehído/metabolismo , Animales , Sitios de Unión , Reactivos de Enlaces Cruzados , Dimerización , Electroforesis en Gel de Poliacrilamida , Cinética , Lactococcus lactis/enzimología , Hígado/enzimología , NADP/metabolismo , Oxidación-Reducción , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Ovinos , Espectrometría de Fluorescencia , Trypanosoma brucei brucei/enzimología , o-Ftalaldehído/farmacologíaRESUMEN
BACKGROUND AND OBJECTIVE: Human leukemic K562 cells are able to undergo erythroid differentiation in vitro when cultured with a variety of inducers, leading to increased expression of embryo-fetal globin genes such as the zita, epsilon and gamma-globin genes. Therefore the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutical potential of new differentiating compounds as well as for studying the molecular mechanism(s) that regulate changes in the expression of embryonic and fetal human globin genes. In this study we explored whether nucleoside triphosphates and related compounds are able to induce differentiation of K562 cells. METHODS: K562 cell differentiation was studied using the benzidine test; hemoglobins were characterized by cellulose acetate gel electrophoresis and mRNA accumulation was investigated by Northern blot analysis. RESULTS: The main conclusion of this paper is that guanine, guanosine and guanine ribonucleotides are effective inducers of K562 cell differentiation. Expression of both Hb Portland and Hb Gower 1 is increased in GTP-induced K562 cells. This increase is associated with greater gamma-globin mRNA accumulation. By contrast, ATP, CTP and UTP are not able to induce erythroid differentiation. INTERPRETATION AND CONCLUSIONS: These findings suggest that guanine, guanosine and guanine ribonucleotides are inducers of erythroid differentiation of K562 cells. This is of some relevance since differentiating compounds have been proposed as antitumor agents. In addition, inducers of erythroid differentiation that stimulate gamma-globin synthesis might be considered in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.
Asunto(s)
Eritrocitos/patología , Nucleótidos de Guanina/farmacología , Guanina/farmacología , Guanosina/farmacología , Leucemia/patología , Diferenciación Celular/efectos de los fármacos , Humanos , Células Tumorales CultivadasRESUMEN
Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.
Asunto(s)
Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Microglía/metabolismo , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/fisiología , Células Cultivadas , HumanosRESUMEN
The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Tripanocidas/farmacología , Trypanosoma brucei brucei/enzimología , Animales , Arsenicales/farmacología , Concentración de Iones de Hidrógeno , Cinética , Hígado/enzimología , Melarsoprol/farmacología , Ovinos , Especificidad de la Especie , Suramina/análogos & derivados , Suramina/farmacologíaRESUMEN
We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.
Asunto(s)
Adenosina Trifosfato/fisiología , Canales de Calcio/efectos de los fármacos , Calcio/fisiología , Activación del Canal Iónico/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Fitohemaglutininas/farmacología , Receptores Purinérgicos P2/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Canales de Calcio/fisiología , Compartimento Celular , Humanos , Potenciales de la Membrana/efectos de los fármacos , Muromonab-CD3/farmacología , Receptores Purinérgicos P2/fisiología , Canales de Sodio/fisiología , Linfocitos T/inmunologíaRESUMEN
Bromopyruvate inactivates 6-phosphogluconate dehydrogenase by affinity labeling. Kinetic analyses, stoichiometry and isolation of a single labelled tryptic peptide of the modified protein indicate that inactivation is due to the affinity labeling of a single cysteine residue, identified as cysteine 401. It thus appears that this cysteine is within a short distance from the protein site involved in the binding of the carboxylate group of the substrate. These results suggest that the carboxylate binding site of proteins could be used as an anchorage point for affinity labeling, and that bromopyruvate can be used to individuate an amino acid residue within few A from this site.
Asunto(s)
Marcadores de Afinidad , Hígado/enzimología , Fosfogluconato Deshidrogenasa/química , Piruvatos , Animales , Sitios de Unión , Cisteína/química , OvinosRESUMEN
Lamb liver phosphogluconate dehydrogenase is inactivated and selectively cleaved during irradiation in the presence of vanadate. Under our experimental conditions, the correlation between the species of vanadate in solution and rates of enzyme inactivation and cleavage indicates tetravanadate as the most likely photosensitizing agent, in agreement with previous data on other proteins. The enzyme is inactivated more rapidly at acidic pH and is partially protected by the coenzyme NADP, but not by the substrate phosphogluconate. Complete inactivation is obtained when only half of the protein is cleaved into smaller peptides. Differences in the pattern of the peptides produced are observed when irradiation is carried out in phosphate rather than in Hepes buffer: in the former instance cleavage results into formation of a main peptide of 47 kDa, while in latter case two additional peptides of 31 and 25 kDa are produced.
Asunto(s)
Hígado/enzimología , Fosfogluconato Deshidrogenasa/metabolismo , Rayos Ultravioleta , Vanadatos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Cinética , Sustancias Macromoleculares , Fragmentos de Péptidos/aislamiento & purificación , Fosfogluconato Deshidrogenasa/efectos de los fármacos , Fosfogluconato Deshidrogenasa/efectos de la radiación , OvinosRESUMEN
We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.
Asunto(s)
Adenosina Trifosfato/fisiología , Espacio Extracelular/metabolismo , Linfocitos/metabolismo , Distrofias Musculares/metabolismo , Línea Celular Transformada , Senescencia Celular , Humanos , Canales Iónicos/metabolismo , Iones , Distrofias Musculares/patología , Valores de ReferenciaRESUMEN
It has been proposed that extracellular ATP (ATPo) may function as a cytotoxic molecule in Ca(2+)-independent cell-mediated lysis by activating plasma membrane P2 purinergic receptors. In the present study the involvement of the P2z purinergic receptor in ATPo as well as cell-mediated lysis was investigated by using the J774 mouse macrophage cell line, which expresses this receptor, and a panel of J774-derived mutant cell clones selected for the lack of P2z receptor activity. We confirmed that the P2z receptor in J774 is associated with ATPo-induced colloido-osmotic lysis but not with apoptosis. Furthermore, we observed that the lack or the inhibition of the P2z purinergic receptor does not affect lytic activity mediated by different types of cytotoxic cell populations. These results on the whole indicate that the P2z receptor is involved in cell membrane damage induced by ATPo but not in cell-mediated cytotoxicity.
Asunto(s)
Adenosina Trifosfato/inmunología , Citotoxicidad Inmunológica/fisiología , Macrófagos/inmunología , Macrófagos/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Línea Celular , ADN/metabolismo , Resistencia a Medicamentos , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2/metabolismo , Linfocitos T Citotóxicos/inmunologíaRESUMEN
In order to find a new reagent for the affinity labelling, 6-phosphogluconate dehydrogenase was treated with glyoxylate, a versatile metabolite with a carboxyl and a reactive aldehydic group. High concentrations of glyoxylate inhibit the enzyme, while in the presence of the reducing agent cyanoborohydride, the enzyme is irreversibly inactivated by only millimolar glyoxylate. This indicates the formation of a Schiff base between the aldehydic group of glyoxylate and one enzyme lysine residue. The kinetics and substrate competition suggest that inactivation is due to affinity labelling. In the first step the inhibitor carboxylic group binds to the substrate carboxyl binding site, and in the second slower step the aldehydic group binds a nearby lysine. We have also found that other enzymes are inactivated by the combined actions of glyoxylate and cyanoborohydride, with a saturation kinetics. Hence, glyoxylate can be helpful to identify specific lysines at the carboxyl binding sites in proteins.
Asunto(s)
Marcadores de Afinidad/metabolismo , Glioxilatos/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Animales , Sitios de Unión , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , OvinosRESUMEN
When cultured in liquid media, samples of the yeast Trichosporon grow readily and degrade phenol; glutamate was found to stimulate both fungal growth and phenol catabolism, with a distinctive lag. In addition, this same strain grows in the presence of 2-chloro-phenol and 2-methyl, 4-chlorophenol (which are also degraded) and in the presence of 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, which are either degraded or not, as in the case of 4-nitrophenol. The kinetics of both growth and of aromatic catabolism is suggestive of inductive phenomena for key metabolic enzymes.
Asunto(s)
Fenoles/metabolismo , Trichosporon/metabolismo , Medios de Cultivo , Glutamatos/farmacología , Ácido Glutámico , Técnicas Microbiológicas , Trichosporon/crecimiento & desarrollo , Trichosporon/aislamiento & purificaciónRESUMEN
The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor.