RESUMEN
Our objective was to investigate the levels of chemokines (MIP1-alpha, MCP-1, and Gro-alpha), Interleukin-18 (IL-18), and Interleukin (IL-6) in bronchoalveolar lavage (BAL) fluid and serum at the onset and ongoing states of sepsis as defined by the American College of Chest Physicians/Society of Critical Care Medicine in septic surgical ICU patients. Our summary background data was to understand the significance of compartmentalized inflammatory mediator production in an immunologically active organ (lung) in comparison with levels in the systemic circulation. The study group consisted of 20 septic patients and 10 non-septic patients on surgical ICU. At the onset of sepsis, both BAL fluid and serum samples were taken and levels of MIP-1alpha, MCP-1, GRO-alpha, IL-18, and IL-6 were measured by ELISA. Furthermore, over a subsequent 8-day period, levels of these mediators were determined in serum. In some experiments, IL-18 mRNA levels were determined in peripheral blood lymphocytes (PBL) of septic and non-septic patients. At the onset of sepsis, MIP-1alpha, MCP-1, GRO-alpha, IL-18, and IL-6 levels were significantly up-regulated in BAL fluid as compared with non-septic controls. In marked contrast, with the exception of IL-18 mRNA and IL-6 peptide, there was no increase in serum levels of inflammatory mediators determined both at the onset and during the ongoing states of sepsis. Based on the present data, monitoring levels of serum chemokines and IL-18 protein as markers of sepsis might be misleading since despite their non-detection in serum, they were highly up-regulated in the lung tissue compartment. These data might underscore the role of MIP-1alpha, MCP-1, GRO-alpha, and IL-18 in the mediation of local tissue damage. Furthermore, these findings raise the notion that mediator measurement in immunologically active organs might serve as pivotal indicators of sepsis prior to the actual fulfillment of specific clinical criteria that defines the patient as being septic.
Asunto(s)
Líquido del Lavado Bronquioalveolar , Quimiocinas/metabolismo , Cuidados Críticos , Interleucina-18/metabolismo , Sepsis/metabolismo , Estudios de Casos y Controles , Quimiocinas/sangre , Quimiocinas/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-18/sangre , Interleucina-18/genética , Interleucina-6/metabolismo , Complicaciones Posoperatorias , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
The effect of acetyl - tyrosyl-valyl-alanyl-aspartyl - chloromethylketone (ac-YVAD-cmk), an irreversible caspase-1 (IL-1beta converting enzyme, ICE) inhibitor on mortality, leukocyte and platelet counts and cytokine levels was investigated in a double-blind rat model of endotoxaemia. Intravenous (i.v.) bolus administration of lipopolysaccharide (LPS) (25-75 mg kg(-1), n=12 per group) to anaesthetized rats induced a dose dependent increase in mortality over 8 h (LD(50)=48 mg kg(-1)). During this period, animals became leukopenic and thrombocytopenic. Serum levels of IL-beta, IL-6, and TNF-alpha were highly elevated. Pretreatment of rats with ac-YVAD-cmk at a dose of 12.5 micromol kg(-1) significantly reduced mortality from 83 to 33% using Log Rank analysis. However, ac-YVAD-cmk did not modify blood cell counts or cytokine profiles as compared with the LPS-drug vehicle group. These data lay credence to the potential importance of caspase-1-inhibition in modifying the inflammatory response to endotoxin. Further investigations are warranted in understanding the relationship between caspase-1 inhibition, cytokine production and animal survival in different experimental paradigms of sepsis.
Asunto(s)
Clorometilcetonas de Aminoácidos/uso terapéutico , Inhibidores de Caspasas , Endotoxemia/prevención & control , Lipopolisacáridos/toxicidad , Animales , Inhibidores de Cisteína Proteinasa/uso terapéutico , Citocinas/sangre , Endotoxemia/sangre , Endotoxemia/inducido químicamente , Leucopenia/etiología , Masculino , Ratas , Ratas Sprague-Dawley , Trombocitopenia/etiologíaRESUMEN
Forty-one patients were analyzed after surgical treatment of Achilles tendon ruptures. The following parameters served as the outcome measure: (1) duration of wearing cast, (2) length of hospital stay, (3) outpatient treatment, (4) time of absence from work, (5) complications, (6) re-rupture rate, (7) subjective evaluation by patients, (8) scar condition, (9) ability to stand on tiptoes, (10) Thompson test, (11) movement of talocrural joint, (12) circumference data of lower extremity, (13) radiographs, (14) power measurement of the ankle (in kg), (15) ultrasound examination, (16) blood cholesterol levels, (17) scoring by Trillat's score. Surgical treatment achieved an excellent or good outcome in 91% of patients as evidenced by the Trillat score. Furthermore, cholesterol levels were found to be elevated in 83% of patients. Given the good results, surgical treatment of Achilles tendon ruptures is recommended, but patients of status post-Achilles tendon rupture should be checked for high cholesterol levels. In the future, controlled, prospective trials need to prove a correlation between Achilles tendon rupture and a pathological blood lipid status.
Asunto(s)
Tendón Calcáneo/lesiones , Tendón Calcáneo/cirugía , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/etiología , Tendón Calcáneo/diagnóstico por imagen , Adulto , Anciano , Colesterol/sangre , Femenino , Estudios de Seguimiento , Humanos , Hipercolesterolemia/sangre , Masculino , Persona de Mediana Edad , Procedimientos de Cirugía Plástica/métodos , Recurrencia , Reoperación , Estudios Retrospectivos , Medición de Riesgo , Rotura/sangre , Rotura/complicaciones , Rotura/diagnóstico , Rotura/cirugía , UltrasonografíaRESUMEN
BACKGROUND: Using differential display reverse transcriptase-polymerase chain reaction we have recently identified mob-1, the novel rat homologue of the human alpha-chemokine IP-10, as a highly inducible gene in adult respiratory distress syndrome (ARDS) lungs. The present study aimed to further implicate mob-1 in the pathogenesis of ARDS. METHODS: Pulmonary mob-1 mRNA up-regulation was confirmed by Northern blot analysis in three different rat models of ARDS-like lung injury and localized to pulmonary macrophages by using in situ hybridization. Also, Escherichia coli-derived recombinant mob-1 (rmob-1) was tested for its properties in relationship to lung injury. RESULTS: In vivo, intratracheal injection of rmob-1 (50 micrograms/rat) induced pulmonary leukosequestration (myeloperoxidase +93% +/- 8% versus control, p < 0.05) with preferential accumulation of neutrophils in bronchoalveolar lavage fluid (36.0% +/- 1.0% versus 0.1% +/- 0.1% in controls, p < 0.01). In vitro, transwell migration studies demonstrated chemotactic activity of rmob-1 (50 to 100 ng/ml) toward human monocytes (+151% +/- 34% versus rmob-1 vehicle, p < 0.01) and only weak chemotaxis for human neutrophils (+15% +/- 0% versus rmob-1 vehicle, p < 0.01). Utilizing a rat aortic ring model ex vivo, rmob-1 at 100 ng/ml exerted a very potent inhibitory effect on angiogenesis (-78.7% +/- 6.3% versus rmob-1 vehicle, p < 0.01), a major component of the resolution phase of ARDS. CONCLUSIONS: Taken together, these data support the involvement of mob-1 in the pathogenic mechanisms of ARDS possibly through chemotaclic actions on inflammatory cells and modulation of angiogenesis in the recovery phase of the disease.
Asunto(s)
Quimiocinas CXC , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Pulmón/inmunología , Síndrome de Dificultad Respiratoria/inmunología , Transcripción Genética , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/citología , Quimiocina CXCL10 , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Clonación Molecular , Citocinas/toxicidad , Cartilla de ADN , Modelos Animales de Enfermedad , Escherichia coli , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Técnicas In Vitro , Pulmón/patología , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/toxicidad , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
The present study investigated the effect of liposome-encapsulated hemoglobin (LEH), an experimental oxygen-carrying resuscitation fluid, on triglyceride, total cholesterol, and low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol measurements. In vivo, the intravenous infusion of LEH (5.6 mL/kg, n = 6) elevated serum triglycerides (+92% vs. baseline, P < .05), total cholesterol (+25% vs. baseline, P < .01), LDL cholesterol (+72% vs. baseline, P < .01) and had no effect on serum HDL cholesterol. In addition, LEH did not alter the elevation in serum triglycerides (+302% vs. baseline, P < .01) and LDL cholesterol (+86% vs. baseline, P < .01) induced by lipopolysaccharide (3.6 mg/kg, i.v., n = 6. Ex vivo, measurements of triglycerides and total cholesterol as well as LDL and HDL cholesterol in whole blood from naive rats were not changed by the addition of LEH (0-50%, n = 6). In vitro, the addition of a fixed concentration of LEH (50%, n = 6) to varying concentrations of cholesterol solution (0-50%), or vice versa, had no effect on cholesterol determination. It is therefore concluded that LEH only minimally affects serum levels of triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol and does not interfere with their measurement.
Asunto(s)
Sustitutos Sanguíneos/farmacología , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Colesterol/sangre , Hemoglobinas/farmacología , Liposomas , Triglicéridos/sangre , Animales , Sustitutos Sanguíneos/administración & dosificación , Hemoglobinas/administración & dosificación , Técnicas In Vitro , Infusiones Intravenosas , Lipopolisacáridos/farmacología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Interferon-gamma inducible protein 10 kD (IP-10) is a highly inducible, primary response gene that belongs to the C-X-C chemokine superfamily. Despite the original cloning of IP-10 in 1985, its biological functions are still unclear although accumulating reports indicate that it is a pleiotropic molecule capable of eliciting potent biological effects, including stimulation of monocytes, natural killer and T-cell migration, regulation of T-cell and bone marrow progenitor maturation, modulation of adhesion molecule expression as well as inhibition of angiogenesis. More interest is now likely to be focused on IP-10 due to the recent cloning of an IP-10 receptor. This paper aims to highlight our current knowledge of IP-10 and its homologues as well as defining its likely involvement in regulating fibroproliferation following inflammatory lung injury.
Asunto(s)
Quimiocinas CXC , Quimiocinas/genética , Quimiocinas/inmunología , Interferón gamma/farmacología , Secuencia de Aminoácidos , Animales , Quimiocina CXCL10 , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoAsunto(s)
Interleucina-2/farmacología , Síndrome de Dificultad Respiratoria/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citocinas/química , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/patología , Microcirculación/efectos de los fármacos , Datos de Secuencia Molecular , Factor de Activación Plaquetaria/farmacología , Circulación Pulmonar/efectos de los fármacos , ARN Mensajero/metabolismo , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/patología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Interleukin (IL)-2-induced microvascular lung injury is an experimental paradigm commonly used to investigate the pathogenesis of the adult respiratory distress syndrome. Since tumor necrosis factor-alpha (TNF-alpha) is known to induce such an injury in vivo and since TNF-alpha is involved in other models of lung injury, we postulated that it might also mediate pulmonary toxicity after IL-2 administration. The present study tested this hypothesis by evaluating the effect of TNF-alpha inhibition on IL-2-induced lung injury in the rat. Recombinant human IL-2 (10(6) U IV per rat, n = 6) elevated lung water, myeloperoxidase activity, and protein accumulation in bronchoalveolar lavage fluid and induced tissue hypoxia. Also, IL-2 enhanced lung tissue TNF-alpha mRNA and peptide (1543 +/- 496 pg/g lung wet weight) localized to alveolar macrophages by in situ hybridization. In marked contrast, IL-2 failed to affect serum TNF-alpha, which remained at undetectable levels. Pretreatment with anti-TNF-alpha monoclonal antibody (25 mg/kg IV, n = 7) or the TNF-alpha synthesis inhibitor rolipram (200 micrograms/kg IV, n = 7) attenuated lung injury and reverted tissue hypoxia. Furthermore, TNF-alpha inhibition prevented the upregulation of lung tissue IL-1 beta, IL-6, cytokine-induced neutrophil chemoattractant, and E-selectin (ELAM-1) but not intercellular adhesion molecule-1 mRNAs in response to IL-2. These data imply that locally produced TNF-alpha mediates IL-2-induced lung inflammation and tissue injury and point to the potential utilization of TNF-alpha inhibitors in treating the pulmonary toxicity of IL-2 immunotherapy.
Asunto(s)
Interleucina-2/toxicidad , Pulmón/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Cricetinae , Perros , Humanos , Interleucina-2/metabolismo , Pulmón/metabolismo , Masculino , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Rolipram , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The original notion that the brain represented an "immune-privileged" organ lacking the capability to produce an inflammatory response to an injury, would appear no longer tenable. Indeed, accumulating evidence during the last decade has shown that the CNS can mount a well-defined inflammatory response to a variety of insults including trauma, ischemia, transplantation, viral infections, toxins as well as neurodegenerative processes. Many aspects of this centrally-derived inflammatory response parallel, to some extent, the nature of such a reaction in the periphery. Through the recent application of molecular biological techniques, new concepts are rapidly emerging as to the molecular mechanisms associated with the development of brain injury. In particular, the importance of cytokines, especially TNF alpha and IL-1 beta, as well as adhesion molecules, has been emphasized in the propagation and maintenance of a CNS inflammatory response. This review will summarize recent observations as to the involvement of these inflammatory mediators in CNS injury and lay claim to the possibility that inhibitors of peripheral inflammation may also be of benefit in treating CNS injuries such as stroke, head trauma, Alzheimer's disease and multiple sclerosis.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Citocinas/fisiología , Animales , Inflamación/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Airway acid aspiration leads to severe microvascular lung injury and pulmonary edema. Recent studies have demonstrated that other conditions associated with microvascular injury such as sepsis and burns can be effectively treated with low-volume hypertonic saline (HTS). Thus, the present study aimed to test whether HTS attenuates aspiration-induced lung injury in the rat. Intratracheal administration of 0.2 ml of 0.1 N HCl (n = 7) induced pulmonary leukosequestration [myeloperoxidase (MPO) activity +446 +/- 34%, P < 0.05; bronchoalveolar lavage (BAL) fluid neutrophil count + 178 +/- 23%, P < 0.05], edema (division 43 +/- 6%, P < 0.01), and microvascular permeability defect (BAL protein concentration +675 +/- 34%, P < 0.01). These changes were associated with tissue hypoxia (skeletal muscle PO2, 49 +/- 8 mm Hg, P < 0.05) and elevated serum TNF alpha (750 +/- 38 pg/ml, P < 0.01). HTS (2400 mosmole/liter) at 5 ml/kg, administered 20 min after aspiration (n = 7), reduced lung pulmonary edema by 58 +/- 7% (P < 0.05) and improved tissue oxygen tension (PO2, 85 +/- 7 mm Hg, P < 0.05) but failed to alter lung MPO and BAL fluid protein and leukocyte count response. Also, HTS did not reduce TNF alpha response to aspiration. These data point to a potential therapeutic role for low-volume HTS in treating aspiration-induced lung injury. In addition, our data suggest that HTS is acting by rapidly shifting fluid from the pulmonary interstitium to the intravascular compartment because it did not inhibit the inflammatory response to aspiration.
Asunto(s)
Neumonía por Aspiración/complicaciones , Edema Pulmonar/tratamiento farmacológico , Edema Pulmonar/etiología , Solución Salina Hipertónica/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar/citología , Ácido Clorhídrico , Recuento de Leucocitos , Pulmón/metabolismo , Oxígeno/metabolismo , Peroxidasa/metabolismo , Neumonía por Aspiración/inducido químicamente , Edema Pulmonar/patología , Ratas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have recently established an animal model of adult respiratory distress syndrome (ARDS)-like microvascular lung injury elicited by infusion of human interleukin-2 (IL-2). Based on the pronounced, transcriptional upregulation of multiple pro-inflammatory mediators in IL-2-induced ARDS, differential display was applied to search for potentially novel genes in this paradigm of lung injury. Differential display on total lung RNA derived from IL-2-challenged rats presented a highly reproducible 3'-UTR fragment profile in which a band (approximately 250 bp), termed B1, was strongly induced. B1 cDNA sequence exhibited 99.14% homology to the 3'-UTR of mob-1, a recently cloned gene belonging to the C-X-C chemokine superfamily. Furthermore, Northern blot analysis showed that IL-2-induced pulmonary mob-1 mRNA was expressed at time points before the onset of lung injury and suppressed after TNF-alpha inhibition. These data imply that lung mob-1 is a novel, highly inducible gene in a clinically relevant model of ARDS and, based on its identification as a chemokine, could participate in the development of lung injury.
Asunto(s)
Quimiocinas CXC , Citocinas/metabolismo , Interleucina-2 , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Quimiocina CXCL10 , Cricetinae , Citocinas/genética , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-2/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Sondas Moleculares/genética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes , Factores de TiempoAsunto(s)
Lesiones Encefálicas/fisiopatología , Inflamación/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Lesiones Encefálicas/prevención & control , Calcio/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/fisiología , Muerte Celular/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Leucocitos/fisiología , Naftalenos/uso terapéutico , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Pirrolidinonas/uso terapéutico , Rolipram , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
OBJECTIVES: To examine the role of complement in the development of acid aspiration-induced lung injury in the rat. It was postulated that inhibition or depletion of complement attenuates aspiration-induced lung injury. DESIGN: Controlled animal trial. SETTING: Animal Laboratory, Jefferson Medical College, Philadelphia, PA. SUBJECTS: Anesthetized rats. INTERVENTIONS: Aspiration was induced by the intratracheal administration of 0.2 mL of 0.1 N hydrochloric acid (n = 7) and lung injury was evaluated by determining water content, myeloperoxidase activity, protein concentration, and leukocyte count in bronchoalveolar lavage fluid. Muscle PO2 was directly measured using a thin-film chamber oxygen sensor and serum tumor necrosis factor-alpha was assayed by enzyme-linked immunosorbent assay. The effect of complement inhibition by recombinant human soluble complement receptor type 1 (n = 8) or complement depletion by cobra venom factor (n = 7) on lung injury was evaluated. MEASUREMENTS AND MAIN RESULTS: Acid aspiration induced pulmonary leukosequestration, edema, and a microvascular permeability defect, along with tissue hypoxia. Pretreatment with soluble complement receptor type 1 (complement inhibition) or cobra venom factor (complement depletion) significantly reduced lung edema (-61 +/- 7%; p < .05), eliminated protein accumulation in bronchoalveolar lavage fluid (p < .01), and improved (p < .05) tissue oxygenation. In contrast, there was no effect of soluble complement receptor type 1 or of cobra venom factor on leukosequestration. CONCLUSIONS: Acid aspiration induces lung injury through a complement-dependent mechanism that leads to microvascular permeability defects. Therefore, the possibility that complement inhibitors may have a salutary effect in humans with aspiration-induced lung injury should be investigated.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/fisiología , Venenos Elapídicos/farmacología , Neumonía por Aspiración/inmunología , Receptores de Complemento/fisiología , Animales , Permeabilidad Capilar , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ácido Clorhídrico , Masculino , Neumonía por Aspiración/inducido químicamente , Neumonía por Aspiración/tratamiento farmacológico , Premedicación , Ratas , Ratas Sprague-Dawley , Proteínas RecombinantesAsunto(s)
Lipopolisacáridos/toxicidad , Pulmón/irrigación sanguínea , Factor de Activación Plaquetaria/toxicidad , Síndrome de Dificultad Respiratoria/etiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos Monoclonales , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Pulmón/patología , Pulmón/ultraestructura , ARN Mensajero/sangre , Ratas , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Acetyl- and butyrylcholinesterases (AChE, BuChE) from various species differ in their substrate specificities and sensitivities to a wide range of inhibitors, yet display conserved sequence, structure and catalytic properties. To determine features that confer these selective properties, residues 58 through 133 of recombinant human BuChE were replaced with the corresponding sequence from human AChE. The replaced region (> 60% identity) spans the Asp70 residue, important for ligand interactions, and the choline binding site, and introduces differences of charge and hydrophobicity in the outer rim and on the surface of the active site gorge. Expressed in microinjected Xenopus laevis oocytes, the resultant chimera retained the catalytic activity, substrate specificity and the Km value toward butyrylthiocholine characteristic of BuChE. Further, it did not acquire substrate inhibition, which is unique to AChE, although it lost the property of substrate activation, characteristic of BuChE. Moreover, the chimera resembled BuChE in its sensitivity to succinylcholine and physostigmine, but acquired the AChE-like sensitivity to echothiophate and iso-OMPA, and displayed an intermediate pattern of inhibition, more similar to that of AChE than of BuChE, toward bambuterol, dibucaine and BW284C51. These findings demonstrate that the exchanged residues are involved in inhibitor recognition, but not in substrate distinction and in direct catalysis. Furthermore, substrate interaction with the exchanged domain may mediate structural changes leading to substrate activation in BuChE and inhibition in AChE. The two AChE-specific aromatic tyrosine residues positioned near Asp70 within this region are hence implicated in the peripheral anionic site of cholinesterases, which is involved in the recognition of various ligands.
Asunto(s)
Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/metabolismo , Acetilcolinesterasa/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Butirilcolinesterasa/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Activación Enzimática , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos , Conformación Proteica , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Xenopus laevisRESUMEN
1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. Thus, it was found that asp70-->gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425-->Pro with gly70 gives rise to the "atypical" butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.
Asunto(s)
Colinesterasas/metabolismo , Parasimpaticomiméticos/farmacología , Animales , Colinesterasas/genética , Humanos , Relación Estructura-ActividadRESUMEN
Acetyl- and butyrylcholinesterase (ACHE, BCHE) from evolutionarily distant species display a high degree of primary sequence homology and have biochemically similar catalytic properties, yet they differ in substrate specificity and affinity for various inhibitors. The biochemical information derived from analyses of ACHE and BCHE from human, Torpedo, mouse, and Drosophila, as well as that from the recombinant forms of their natural variants and site-directed mutants, can currently be re-examined in view of the recent X-ray crystallography data revealing the three-dimensional structure of Torpedo ACHE. The picture that emerges deepens the insight into the biochemical basis for choline ester catalysis and the complex mechanism of interaction between cholinesterases and their numerous ligands.
Asunto(s)
Acetilcolinesterasa/química , Butirilcolinesterasa/química , Animales , Sitios de Unión , Humanos , Ligandos , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.
Asunto(s)
Butirilcolinesterasa/genética , Colinesterasas/genética , Variación Genética , Mutagénesis Sitio-Dirigida , Oocitos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Butirilcolinesterasa/aislamiento & purificación , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Colinesterasas/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Conformación Proteica , Ingeniería de Proteínas , ARN/genética , ARN/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Especificidad por Sustrato , XenopusRESUMEN
Structure-function relationships of recombinant human butyrylcholinesterase (CHE) variants were investigated by Xenopus oocyte microinjection. A Ser-425 to Pro-425 mutation failed to modify ligand binding properties. In contrast, Asp-70 to Gly-70 substitution significantly reduced CHE binding capacity for succinylcholine and specific inhibitors, demonstrating Asp-70 as a key anionic site component for certain ligands. Furthermore, the presence of both mutations rendered CHE totally resistant to succinylcholine and dibucaine inhibition, while all mutant proteins bound butyrylthiocholine, benzoylcholine, and propionylcholine normally. These findings imply structural interactions between the conserved Asp-70 and Ser-425 regions in cholinesterases and suggest the contribution of additional electronegative amino acids to anionic site binding.