RESUMEN
When Saccharomyces carlsbergensis G-517 was grown in 10 mM galactose as the carbon source, the addition of 2-deoxy-D-glucose restricted the uptake of galactose, [3H]uridine and [3H]leucine, and restricted invertase synthesis (beta-D-fructofuranoside fructohydrolase; EC 3.2.1.26) for a period of 60-90 min. During this time, the radioactive antimetabolite was taken up by the cells; afterwards, invertase synthesis was enhanced, and the utilizaton rate of galactose, [3H]uridine and [3H]leucine increased until it reached that of the control culture. When glucose was used as a carbon source, sugar utilization and uptake of radioactive precursors were unaffected by addition of the deoxysugar.
Asunto(s)
Desoxiazúcares/farmacología , Desoxiglucosa/farmacología , Proteínas Fúngicas/biosíntesis , ARN de Hongos/biosíntesis , Saccharomyces/metabolismo , Cinética , Leucina/metabolismo , Oxiquinolina/farmacología , Saccharomyces/efectos de los fármacos , Sacarasa/biosíntesis , Uridina/metabolismoRESUMEN
A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.
Asunto(s)
Galactosidasas/biosíntesis , Saccharomyces/enzimología , alfa-Galactosidasa/biosíntesis , Carbohidratos/farmacología , Inducción Enzimática/efectos de los fármacos , Cinética , Lactosa/farmacología , Relación Estructura-ActividadRESUMEN
Cholera toxin can alter the ion permeability of brush border membrane vesicles from rabbit small intestine. This alteration is reflected by differences in membrane potential-stimulated, Na-dependent, D-[3H]glucose transport by these vesicles, as well as by an enhancement in the accumulation of the lipophilic cation [3H]tetraphenylphosphonium in response to an artificially imposed membrane potential. Analogous effects were observed when the intact tissue was treated with the toxin and the vesicles subsequently obtained. An important implication of this finding is that cholera toxin does not need to activate adenylate cyclase to induce permeability changes in the cell membrane since the experiments were carried out in conditions where neither ATP nor cyclic AMP was present.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Toxina del Cólera/farmacología , Glucosa/metabolismo , Intestino Delgado/metabolismo , Microvellosidades/metabolismo , Compuestos Organofosforados , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico Activo/efectos de los fármacos , Indicadores y Reactivos/metabolismo , Microvellosidades/efectos de los fármacos , Compuestos Onio/metabolismo , Conejos , Sodio/farmacologíaRESUMEN
A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source.
Asunto(s)
Carboxipeptidasas/genética , ADN de Hongos/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN/análisis , Mapeo RestrictivoRESUMEN
The rate of recovery of rat myocardial alpha 1-adrenoceptor density and responsiveness after in vivo block with phenoxybenzamine (1 mg/kg, i.p.) have been investigated by measuring [3H]prazosin binding, and noradrenaline-stimulated [3H]inositol phosphate production. Repopulation of alpha 1-adrenoceptors was monoexponential, with a t1/2 of 33 h; functional recovery was also monoexponential, with t1/2 of 28 h. Furthermore, our results clearly demonstrate the absence of a receptor reserve for alpha 1-adrenoceptors mediating noradrenaline-stimulated phosphoinositide breakdown in rat myocardial tissue. These observations indicate a close relationship between the density of [3H]prazosin binding sites and the ability of alpha 1-adrenoceptors to respond to noradrenaline. Moreover, based on competition curves for inhibition of specific [3H]prazosin by WB-4101 to rat myocardial membranes 48 h and 7 days after the administration of phenoxybenzamine, the results suggest that rat myocardial membranes contain both alpha 1-adrenoceptors subtypes, i.e., alpha 1A and alpha 1B, in an approximate ratio of 20:80, and this relative ratio does not seem to be altered during the recovery process.
Asunto(s)
Fosfatos de Inositol/metabolismo , Miocardio/metabolismo , Fenoxibenzamina/farmacología , Prazosina/metabolismo , Receptores Adrenérgicos alfa/efectos de los fármacos , Alquilación , Animales , Sitios de Unión , Corazón/efectos de los fármacos , Hidrólisis , Masculino , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/metabolismoRESUMEN
The effects of acute treatment with 1,2,3,4-tetrahydro-9-aminoacridine (THA), a 4-aminopyridine derivative clinically effective in Alzheimer's disease, on beta-adrenoceptor-linked cyclic AMP accumulation have been investigated in cortical and hippocampal structures of young and middle-aged rats. In a first series of experiments, pretreatment with 2.5 mg/kg THA decreased basal cyclic AMP accumulation. When a phosphodiesterase inhibitor was added to the preparation, THA again decreased cyclic AMP levels in young rats, but failed to significantly modify cyclic AMP accumulation in middle-aged animals. Finally, in isoprenaline-stimulated conditions, acute treatment with tacrine was able to diminish cyclic AMP accumulation in every group of rats. It is suggested that the neurochemical action of THA in mammalian brain is more complex than earlier has been anticipated and may involve an action on beta-adrenoceptors.
Asunto(s)
Encéfalo/metabolismo , AMP Cíclico/metabolismo , Isoproterenol/farmacología , Receptores Adrenérgicos beta/fisiología , Tacrina/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Envejecimiento , Animales , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Inhibidores de la Colinesterasa/farmacología , Hipocampo/metabolismo , Masculino , Ratas , Ratas Endogámicas , Receptores Adrenérgicos beta/efectos de los fármacos , Valores de ReferenciaRESUMEN
The biochemical mechanisms involved in the alpha 1-adrenoceptor up-regulation and possible changes in subtypes of adrenoceptors in the rat heart after chemical denervation were investigated. The effects of acute 6-hydroxydopamine treatment (two increasing doses 24 h apart) on the pseudo-steady state densities and turnover rates of alpha 1-adrenoceptors were studied in ventricular myocardium of the rat. We have assessed the repopulation kinetics of [3H]prazosin binding sites after irreversible inactivation of alpha 1-adrenoceptors induced by a single dose of phenoxybenzamine (1 mg/kg i.p.) in rats acutely treated either with 6-hydroxy-dopamine or with vehicle (control animals). Seven days after the last administration of 6-hydroxydopamine an enhanced density of [3H]prazosin binding sites (Bmax 58.7 +/- 3.6 fmol/mg protein vehicle-treated rats versus 82.6 +/- 5.3 fmol/mg protein 6-hydroxydopamine-treated rats) was observed. This was not accompanied by changes in the dissociation constant value. Furthermore, the proportion of high affinity sites for WB-4101 was altered (21 +/- 2% versus 72 +/- 3% for animals treated with vehicle and 6-hydroxydopamine, respectively). In rat myocardium, alpha 1-adrenoceptor turnover, evaluated during the 6-hydroxydopamine-induced up-regulation (7-19 days after the completion of treatment with 6-hydroxydopamine) revealed an increase in the half-life of the alpha 1-adrenoceptor (t1/2 of 67.2 h versus 38.7 h in control animals). The present study confirms an increase in alpha 1-adrenoceptors in rat myocardium after chemical denervation and reveals that the effect is almost completely confined to the alpha 1A-adrenoceptor subtype. Furthermore, the up-regulation of alpha 1A-adrenoceptors is the result of a decrease in the cellular processes that control the rate of receptor degradation.
Asunto(s)
Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/metabolismo , Miocardio/metabolismo , Oxidopamina/farmacología , Prazosina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Análisis de Varianza , Animales , Peso Corporal , Dioxanos/metabolismo , Masculino , Norepinefrina/metabolismo , Norepinefrina/fisiología , Tamaño de los Órganos , Fenoxibenzamina/farmacología , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
The tumor promoter okadaic acid (OKA), is a marine toxin of algal origin, identified as a potent inhibitor of protein phosphatases 1 and 2A, and possibly enhancing calcium influx through voltage dependent calcium channels (VSSC). We now report that OKA at concentrations as low as 0.5 nM produced neurotoxicity, characterized first by the desintegration of the neurites and swelling of cell bodies, and later by cellular death. Non-neuronal cells viability and morphology were unaffected up to at least 5 nM OKA. Neurons sensitivity to the toxin changed with age in culture. Maximum neurotoxicity was observed in neurons at 9 DIC, when the OKA concentration producing half of the maximum reduction in neuronal survival (EC50) was approximately 0.65 nM. At 5 DIC or 19 DIC (EC50 approximately 2.5 nM and approximately 4.5 nM respectively), neurons appeared to be less sensitive to OKA. Neurotoxicity by OKA was not reduced by VSCC antagonists such as nifedipine and verapamil, nor by antagonists of excitatory aminoacid (EAA) receptors including APV, MK801 or CNQX. VSCC antagonists and EAA receptors antagonists fully protected from neurotoxicity induced by depolarization with KCl. These results suggest that OKA mechanism of neurotoxicity may not directly involve VSCC, endogenous EAA release and EAA receptors, but may depend upon other neurochemical events.
Asunto(s)
Cerebelo/efectos de los fármacos , Éteres Cíclicos/toxicidad , Neurotoxinas/toxicidad , Animales , Bloqueadores de los Canales de Calcio/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Neuronas/efectos de los fármacos , Ácido Ocadaico , Ratas , Receptores de Neurotransmisores/antagonistas & inhibidoresRESUMEN
The effects of ageing on alpha 1-adrenoceptor subtypes have been examined in heart ventricular muscle of young (2-3 months) and middle-aged (18 months) Sprague-Dawley rats. Radioligand binding studies with [3H]prazosin revealed an age-related loss of binding sites (Bmax 56.7 +/- 1.93 fmol (mg protein)-1 age 2 months vs 31.7 +/- 2.45 fmol (mg protein)-1 age 18 months) not followed by changes in the dissociation constant value (Kd 0.16 +/- 0.03 nM age 2 months and 0.10 +/- 0.03 nM age 18 months). Competition curves with WB 4101 showed two distinct sites with different affinities, the proportion of sites with high affinity being similar for both age groups (22.2 +/- 1.89% vs 17.8 +/- 1.96% for animals aged 2 and 18 months, respectively). Agonist displacement curves of [3H]prazosin indicate the existence of two different affinity sites for the agonist, that are maintained regardless of the ageing process (R(high) = 16.2 +/- 1.54% and R(low) = 83.8 +/- 1.89% in rats aged 2 months and R(high) = 16.3 +/- 3.23% and R(low) = 83.7 +/- 3.95% in rats aged 18 months). The fractional inactivation of alpha 1-adrenoceptors by chloroethylclonidine resulted in a loss of [3H]prazosin specific binding, and a percentage of 22.5 +/- 0.95 and 22.6 +/- 4.2 of remaining binding sites for the groups of 2 and 18 months of age, respectively. The percentage of chloroethylclonidine-insensitive [3H]prazosin binding sites was similar to those with high affinity for WB4101. The present study confirms a decline of alpha 1-adrenoceptors with increasing age and reveals that the equilibrium of the expression of the two existing subpopulations of the receptor is maintained during ageing.
Asunto(s)
Envejecimiento/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas Adrenérgicos alfa/farmacocinética , Alquilantes/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Clonidina/análogos & derivados , Clonidina/farmacología , Dioxanos/farmacocinética , Técnicas In Vitro , Norepinefrina/farmacocinética , Prazosina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The endotoxin that triggers an immune response to Gram-negative bacterial infection namely lipopolysaccharide (LPS), is also associated with gastrointestinal abnormalities and induces the release of proinflammatory cytokines such as IL-1 and TNF-α. The main aim of this study was to determine the effect of cytokine release on intestinal D-fructose absorption in LPS-treated rabbits in order to provide information that could be used to understand their septic status. The results obtained, using whole tissue and brush border membrane vesicles from rabbit jejunum, showed that LPS, TNF-α and IL-1ß inhibit d-fructose absorption across the jejunum. The effect of LPS is completely reversed by a TNF-α antagonist and partially by a specific IL-1 receptor antagonist (IL-1ra) and disappears completely in the presence of both these cytokine antagonists. Similarly, the effects of TNF-α and LPS were not totally blocked by IL-1ra, whereas the effect of IL-1ß disappeared completely in the presence of a TNF antagonist. In summary, these results show that TNF-α and IL-1ß could act synergistically on sugar absorption in rabbit with LPS-induced sepsis. In addition, the effects of IL-1ß depend on, or are related to TNF-α production since this effect returns to basal (control) levels in the presence of a TNF-α antagonist.
Asunto(s)
Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Yeyuno/metabolismo , Lipopolisacáridos/toxicidad , Conejos , Animales , Relación Dosis-Respuesta a Droga , Fructosa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Receptores de Interleucina-1/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/biosíntesis , Receptor trkB/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Apoptosis/genética , Autopsia , Encéfalo/patología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Humanos , Lentivirus , Masculino , Neuronas/metabolismo , Neuronas/patología , Plásmidos , Cultivo Primario de Células , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Receptor trkB/genética , Transducción de Señal/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patologíaAsunto(s)
Cerebelo/efectos de los fármacos , Éteres Cíclicos/toxicidad , Toxinas Marinas/toxicidad , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/patología , Colforsina/farmacología , Glutamatos/toxicidad , Ácido Glutámico , Ácido Kaínico/análogos & derivados , Ácido Kaínico/toxicidad , Modelos Neurológicos , Neuronas/patología , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Ratas , Mariscos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The use of online teaching tools facilitate the incorporation of self-learning methods. With a view to encouraging convergence in teaching tools and methods in Occupational Legal Medicine, an initiative was set up within the classes of Legal and Forensic Medicine at Saragossa University, as part of the EU funded NetWoRM project, which has been led since 1999 by Ludwig-Maximilians-Universität in Munich (Germany). The interest of medical students in Occupational Legal Medicine has so far been low and in addition different aspects complicate the teaching of Occupational Legal Medicine at medical schools: One reason for the low interest is the limited availability of bedside teaching, one of the students' most favourite and effective way to learn. The reason for that is that most medical schools with occupational departments only have outpatient clinics. "Interesting" patients who be need for educational purposes are therefore only available for a limited part of the day. However, in order to recognize and prevent occupational disorders each medical student and physician needs profound clinical knowledge in Occupational Legal Medicine. This project has proven to be highly efficient in permitting the creation and validation of teaching tools which cover and improve the traditional training of the Occupational Legal Medicine programme imparted in the degree of Medicine.
Asunto(s)
Instrucción por Computador , Medicina Legal/educación , Internet , Medicina del Trabajo/educación , Aprendizaje Basado en Problemas , Educación de Postgrado en Medicina , Europa (Continente) , Humanos , Estudiantes de MedicinaRESUMEN
Aggression against healthcare workers is a problem of important consequences which is becoming a focus of research. However, its possible effects on psychological health have not been studied sufficiently in spite of the fact that they may be of importance even in the absence of physical aggression [Winstnaley S, Whittington R. Aggression towards health care staff in a UK general hospital: variation among professions. J Clin Nurs 2004;13:3-10,[1]].
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Agresión/psicología , Personal de Salud/psicología , Ansiedad/epidemiología , Agotamiento Profesional/psicología , Estudios Transversales , Depresión/epidemiología , Humanos , Satisfacción en el Trabajo , Medicina , España/epidemiología , Especialidades QuirúrgicasRESUMEN
The N-methyl-D-aspartate receptor (NMDAR) is central to physiological and pathological functioning of neurons. Although promising results are beginning to be obtained in the treatment of dementias, clinical trials with NMDAR antagonists for stroke, trauma and neurodegenerative disorders, such as Hungtinton's disease, have failed before. In order to design effective therapies to prevent excitotoxic neuronal death, it is critical to characterize the consequences of excessive NMDAR activation on its expression and function. Previous data have reported partial downregulation of the NR1 and NR2B receptor subunits in response to excitotoxicity and cerebral ischemia. However, the effect of NMDAR overactivation on NR2A, a subunit fundamental to synaptic transmission and neuronal survival, is still elusive. In this study, we report the rapid and extensive proteolytic processing of NR2A, together with the scaffolding protein postsynaptic density-95 (PSD-95), induced by excitotoxic stimulation of cortical neurons in vitro and by transient focal cerebral ischemia. Processing of the C terminus of NR2A is irreversibly induced by brief agonist exposure of NR2B-containing receptors, and requires calcium influx and the activity of calpain, also responsible for PSD-95 cleavage. The outcome is a truncated NR2A subunit that is stable and capable to interact with NR1 at the surface of neurons, but lacking the structural domains required for association with scaffolding, downstream signaling and cytoskeletal proteins. Therefore, a rapid and significant uncoupling of synaptic NMDARs from downstream survival pathways is expected to occur during ischemia. This novel mechanism induced by excitotoxicity helps to explain the failure of most therapies based on NMDAR antagonists.
Asunto(s)
Isquemia Encefálica/metabolismo , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Lesiones Encefálicas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Masculino , Proteínas de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
BACKGROUND: The Spanish Family Medicine National Commission is proposing a new portfolio-type Specialist Training Book (STB). OBJECTIVE: To pilot its contents, structure, and implementation strategies. DESIGN: Cross-sectional, descriptive study. SETTING: A Provincial Family Medicine Teaching Unit. PARTICIPANTS: Twenty-eight tutors and 36 residents. METHODS: For 9 months the participants conducted a training assessment on diverse areas of competence by means of tasks at work. Tutors recorded information on the quality of reflection achieved by residents and the tasks they performed by means of the card model proposed in the STB. Residents filled in an ad hoc survey. A univariate analysis of quantitative data was conducted. RESULTS: Thirty-three surveys were received from residents; 21 tutors handed in 67 evaluation reports (average: 3 per tutor). They dealt with all the areas of competence, particularly those of communication, teaching, and ethics. Tasks most used were clinical sessions, critical incidents and video-recording. Both tutors and residents thought that the new method could be useful for reflecting on clinical practice, understanding their own areas of competence better and for strengthening the tutor-trainee relationship, especially if some suggestions to improve its practical use and reduce time and effort involved were taken into account. CONCLUSIONS: The new STB in its current version or with some modifications is a useful tool for residents' training assessment and is probably accepted well in our ambit.