RESUMEN
Spinal cord stimulation is a minimally invasive mode of treatment in the management of certain forms of chronic pain that do not respond to conventional pain therapy. Several authors have reported encouraging findings with this technique. Over a 10-year period in a single centre, 254 patients were subjected to a trial period of spinal cord stimulation with an externalized pulse generator. Two hundred and seventeen of the patients showed satisfactory results justifying permanent implantation of a spinal cord stimulation system. In 1998, an independent physician invited 153 patients (155 pain cases), who still had the system in place and who could be contacted, for an interview. The aim of this study was to evaluate the efficacy of an implanted spinal cord stimulation system in terms of pain relief and quality of life and to assess the accuracy of the patient selection criteria. The results of this study demonstrate a high success rate as evaluated by the patients' own assessments--68% of the patients rated the result of the treatment as excellent to good after an average follow-up of almost 4 years. The resumption of work by 31% of patients who had been working before the onset of pain supports these positive findings.
Asunto(s)
Terapia por Estimulación Eléctrica/estadística & datos numéricos , Clínicas de Dolor/estadística & datos numéricos , Manejo del Dolor , Dolor/psicología , Médula Espinal/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Analgésicos/uso terapéutico , Bélgica , Terapia por Estimulación Eléctrica/efectos adversos , Terapia por Estimulación Eléctrica/instrumentación , Electrodos/normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Profesionales/etiología , Dolor/fisiopatología , Clínicas de Dolor/tendencias , Dimensión del Dolor/métodos , Aceptación de la Atención de Salud/estadística & datos numéricos , Selección de Paciente , Calidad de Vida/psicología , Trastornos del Sueño-Vigilia/etiología , Trastornos del Sueño-Vigilia/fisiopatología , Médula Espinal/fisiopatología , Resultado del TratamientoRESUMEN
Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.
Asunto(s)
Apirasa/aislamiento & purificación , Apirasa/metabolismo , Riñón/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Apirasa/genética , Bovinos , Humanos , Inmunohistoquímica , Técnicas In Vitro , Microscopía Electrónica , Peso Molecular , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos , Distribución TisularRESUMEN
In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.
Asunto(s)
Isoenzimas/metabolismo , Pirofosfatasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Apirasa/metabolismo , Células COS , Clonación Molecular , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Pirofosfatasas/química , Pirofosfatasas/genética , PorcinosRESUMEN
Spinally administered opioids must be a last step in the therapeutical arsenal of chronic benigne pain. It is an invasive technique not free from adverse effects. Two chronic pain patients received an implantable Synchromed pump for treatment with spinal opiates after a trial period of resp. 3.5 and 5.5 months. Due to a misprogrammation (both on the same day) they received very high doses of spinal opiates. This caused relatively few side effects, which did not seem to require immediate treatment. A short time development of tolerance to life threatening side-effects has been proven by this accidental administration of high-dose intraspinal opiates. It is critical that care providers are knowledgeable and well-trained about implantable infusion systems. Programmation and refills must always be performed with care.
Asunto(s)
Analgésicos Opioides/envenenamiento , Falla de Equipo , Bombas de Infusión , Morfina/envenenamiento , Anciano , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/uso terapéutico , Femenino , Humanos , Hipofosfatemia Familiar/complicaciones , Inflamación/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Morfina/administración & dosificación , Morfina/uso terapéutico , Osteoporosis/complicaciones , Dolor Intratable/tratamiento farmacológico , Diafragma PélvicoRESUMEN
1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 microM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5'-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.
Asunto(s)
División Celular/efectos de los fármacos , Nucleósidos/farmacología , Nucleótidos/farmacología , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/metabolismo , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Línea Celular Transformada , Fosfatos de Dinucleósidos/farmacología , Dipiridamol/farmacología , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Levamisol/farmacología , Nucleósido-Trifosfatasa , Suramina/farmacología , Timidina/metabolismo , Triazinas/farmacologíaRESUMEN
A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing 141Ce3+ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5'-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce3+ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.
Asunto(s)
5'-Nucleotidasa/análisis , Adenosina Trifosfatasas/análisis , Apirasa/análisis , Autorradiografía/métodos , Histocitoquímica/métodos , Adenosina/análisis , Animales , Línea Celular , Radioisótopos de Cerio , Haplorrinos , HumanosRESUMEN
The protein responsible for the (Ca2+ or Mg2+)-ATPase activity in brush-border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N-ethylmaleimide-sensitive Mg(2+)-ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5'-triphosphates and inhibitors. Solubilization of the (Ca2+ or Mg2+)-ATPase from brush-border membrane vesicles was accomplished with Nonidet P-40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable. A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet-P-40-solubilized brush borders. A similar degree of purification was achieved by ion-exchange chromatography of dodecylmaltoside-solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg2+)-ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity. Labeling of solubilized brush-border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90 kDa is the most probable candidate for the catalytic peptide of the (Ca2+ or Mg2+)-ATPase. Finally, immunoprecipitation as well as semi-dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90-kDa component is a reasonable candidate for the (Ca2+ or Mg2+)-ATPase in renal brush-border membranes.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Corteza Renal/enzimología , Adenosina Trifosfato/química , Marcadores de Afinidad , Animales , Western Blotting , ATPasa de Ca(2+) y Mg(2+)/química , ATPasa de Ca(2+) y Mg(2+)/aislamiento & purificación , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/aislamiento & purificación , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Microvellosidades/enzimología , Pruebas de Precipitina , PorcinosRESUMEN
Basolateral and brush-border vesicles from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. A low-affinity (Ca2+ or Mg2+)-ATPase which co-migrated with alkaline phosphatase was demonstrated. A considerable enrichment (by a factor of 10) of this ATPase activity was only observed in the brush-border and not in the basolateral membrane fractions. Maximal stimulation of this brush-border enzyme by Ca2+ was achieved when the ratio of Ca2+ to ATP reached a value between 1 and 2. The enzyme was not inhibited by excess Ca2+ or Mg2+. A kinetic analysis of the azide-insensitive (Ca2+ or Mg2+)-ATPase gave a Km of 0.43 mM for Ca-ATP and of 0.14 mM for Mg-ATP.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/análisis , ATPasas Transportadoras de Calcio/análisis , Túbulos Renales Proximales/enzimología , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Membrana Basal/enzimología , ATPasas Transportadoras de Calcio/metabolismo , Corteza Renal/enzimología , Túbulos Renales Proximales/ultraestructura , Cinética , Microscopía Electrónica , Microvellosidades/enzimología , PorcinosRESUMEN
Basal-lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. In each type of membrane, azide-insensitive, low-affinity Ca2+-ATPase and Mg2+-ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPases: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca-ATP or Mg-ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 microM of vanadate, N,N'-dicyclohexylcarbodiimide, e diethylstilbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3-stimulated Mg2+-ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3-stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Azidas/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Corteza Renal/enzimología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/aislamiento & purificación , Animales , ATPasa de Ca(2+) y Mg(2+) , ATPasas Transportadoras de Calcio/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Electroforesis/métodos , Activación Enzimática/efectos de los fármacos , Femenino , Membranas/enzimología , Microvellosidades/enzimología , Fracciones Subcelulares/metabolismo , PorcinosRESUMEN
We have developed a method for the large-scale isolation of active ribosomal subunits from human placenta. The technique involves incubating crude ribosomes for 15 min at 37 degrees C with 0.2 mM puromycin in 50 mM Tris-HCl buffer, pH 7.6, 500 mM KCl and 3 mM MgCl2 followed by centrifugation at 5 degrees C in a BXV zonal rotor using an equivolumetric sucrose gradient in the same buffer, upon which 80--90% of all ribosomes are dissociated into subunits. The purified subunits differ in their chemical composition, the 60-S particle containing no more than 36% protein whereas the 40-S subunit consists of 43% protein. In poly(U)-directed protein synthesis, tested in a completely homologous cell-free system, one recombined couple polymerizes at 37 degrees C 12 to 17 phenylalanine residues at an initial rate of 0.7 residues per minute. However, free 80-S ribosomes obtained by puromycin treatment of the crude ribosomes and reassociation of the subunits without prior isolation, have an even higher incorporating activity (20--25 mol phenylalanine/mol of ribosome). At least 55% of the subunits were estimated to actively participate in the polyphenylalanine synthesis.