Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.

Accions no canoniques dels Inhibidors d'Acetilcolinesterasa (IAChE ): estudis in vivo i in vitro de les huprines i heterodímers huprina X i tacrina

Projecte de recerca elaborat a partir d’una estada al Karolinska Institutet, Suècia, entre febrer i juliol del 2007. Els anticolinesteràsics són els fàrmacs aprovats actualment pel tractament pal•liatiu d’Alzheimer. En l’intent de trobar fàrmacs capaços de modificar l’evolució de la malaltia s’han sintentitzat nous compostos que conserven l’activitat anticolinesteràsica però que a més, són capaços d’interaccionar amb la cascada de formació de la proteïna beta-amiloid i la proteïna tau, principals agents patogènics d’aquesta malaltia. L’objectiu fonamental del projecte era el d’analitzar el contingut de proteina beta-amiloid, de receptors nicotínics, de GPAF i sinaptofisina en mostres de cervell de ratolins triple transgènics tractats previament amb Huprina X i Huperzina A.

Desarrollo de un modelo in vivo murino de isquemia y reperfusión miocárdica

En aquellos pacientes que sufren un infarto de miocardio (IM), entender los procesos causantes de la muerte celular secundaria a la isquemia/reperfusión (I/R) es esencial para desarrollar estrategias capaces de prevenirla. La reciente disponibilidad de cepas de ratones transgénicos convierte al modelo murino con isquemia transitoria en una herramienta de gran interés para el estudio de los procesos causantes de esta muerte. El objetivo de este trabajo experimental ha sido establecer en nuestro laboratorio un modelo de I/R miocárdica in vivo en ratón mediante la oclusión de la arteria coronaria descendente anterior izquierda (ADA) con el fin de poder ser utilizado en futuros protocolos experimentales usando ratones transgénicos disponibles en el laboratorio.

Influence of the in vivo deregulation of the expression of ntrk3 (trkc) on cortical development of a murine model of panic/anxiety disorder

Alteracions durant el desenvolupament cerebral produirien canvis en la connectivitat neuronal i la bioquímica cel•lular que podrien resultar en una disfunció cognitiva i/o emocional, desembocant a trastorns psiquiàtrics. Les neurotrofines intervenen en els processos del neurodesenvolupament i en la funcionalitat del cervell adult i, conseqüentment, serien bons candidats com a factors de predisposició en diverses malalties mentals. S’ha suggerit la implicació del receptor de la neurotrofina 3, TrkC, en el trastorn de pànic. Nosaltres proposem que la sobreexpressió del gen NTRK3 (TrkC) és un mediador comú dels desencadenants genètics i ambientals d’aquest trastorn. Concretament, la seva desregulació podria produir canvis estructurals i funcionals a l’escorça cerebral dels pacients pel seu paper durant l’establiment dels circuïts corticals i la neuroplasticitat a l’adult, probablement esdevenint elements de predisposició a patir atacs de pànic. Els objectius principals d’aquest treball han estat: 1/determinar la contribució específica del gen NTRK3 a les alteracions de l’escorça cerebral observades en pacients, utilitzant un model murí modificat genèticament (TgNTRK3), i 2/analitzar l’impacte específic de la sobreexpressió de NTRK3 sobre la corticogènesi durant estadis embrionaris o postnatals estudiant la neurogènesi i la neuritogènesi. Els resultats indiquen que la sobreexpressió de NTRK3 als ratolins produeix una reducció del gruix de l’escorça frontal, recapitulant la hipofrontalitat dels pacients, que comportaria una menor inhibició dels nuclis subcorticals del sistema límbic com l’amígdala, i alteracions citoarquitectòniques a l’escorça prefrontal medial que recolzen la hipòtesi del seu mal funcionament. Tanmateix, els ratolins TgNTRK3 presenten canvis estructurals a l’escorça somatosensorial, suggerint que el processament de la informació sensorial podria estar alterat, el que encara no s’ha explorat en pacients. La sobreexpressió de NTRK3 també afecta la neuritogènesi en cultius primaris corticals i modifica la resposta de les neurones a l’estimulació amb neurotrofines. Per tant, el fenotip cortical adult dels TgNTRK3 podria dependre d’alteracions durant la corticogènesi.

Caracterización funcional de las HERC ubiquitina ligasas en modelos in vivo

La família de proteïnes HERC contenen dos dominis característics: HECT i RLD (RCC1-like). Proteïnes amb dominis HECT funcionen com ubiquitina ligasas i proteïnes amb dominis RLD actuen com a reguladors de GTPases. En humans, la família HERC està formada per sis membres: les gegants (HERC1-2) i les petites (HERC3-6). HERC1 va ser la primera a identificar-se, conté dos dominis RLD (RLD1 i RLD2) i ha estat implicada en tràfic de membrana, proliferació i creixement cel.lular per les seves interaccions amb clatrina, M2-piruvat quinasa i tuberina (TSC2). Aquí es descriu la caracterització del ratolí "rescat" d'una mutació espontània i recessiva anomenada tambaleante, que causa una degeneració progressiva de les cèl.lules de Purkinje amb atàxia severa, un creixement reduït i una menor supervivència. Aquest rescat va ser realitzat amb un transgèn de cDNA de HERC1 humà i demostra que HERC1 és el responsable del fenotip oscil.lant. Hem generats animals knock-outs de HERC1 que no expressen el extremo ubiquitina ligasa de la proteïna, i que no mimetitzen el fenotip tambaleante. La resposta de MEFs de tambaleante a insulina tampoc mimetitza l'activació reduïda dels substrats de mTOR, ni l'autofàgia observats en l'animal tambaleante. Els nostres resultats suggereixen que HERC1 podria estar implicada en la regulació de la biogènesi ribosomal. Aquestes observacions contribueixen a la caracterització funcional de la ubiquitina ligasa HERC1 i demostren un paper fins ara desconegut de HERC1 en creixement i neurodegeneració.

In silico meets in vivo

A report of the 6th Georgia Tech-Oak Ridge National Lab International Conference on Bioinformatics 'In silico Biology: Gene Discovery and Systems Genomics', Atlanta, USA, 15-17 November, 2007.

Patient-specific computational hemodynamics of intracranial aneurysms from 3D rotational angiography and CT angiography: an in vivo reproducibility study

Patient-specific simulations of the hemodynamics in intracranial aneurysms can be constructed by using image-based vascular models and CFD techniques. This work evaluates the impact of the choice of imaging technique on these simulations

Análisis in vivo de la vasculopatía arterial pulmonar mediante ultrasonido intravascular (IVUS) en pacientes con insuficiencia respiratoria crónica evaluados para trasplante pulmonar

El objetivo del estudio es evaluar el remodelado estructural y funcional de las arterias pulmonares asociado a insuficiencia respiratoria crónica severa, mediante ecografía intravascular (IVUS). Se incluyeron 80 pacientes en estudio pretrasplante pulmonar a los que se les realizó cateterismo cardiaco derecho e IVUS de una arteria pulmonar de mediano calibre. A través del IVUS se determinó el módulo elástico, pulsatilidad y porcentaje de fibrosis arterial. La insuficiencia respiratoria crónica se asoció a una vasculopatía arterial pulmonar severa, independientemente de la presencia de hipertensión pulmonar. Los pacientes con EPOC (enfermedad pulmonar obstructiva crónica) presentaban un mayor grado de fibrosis arterial, mientras que los pacientes con EPID (enfermedad pulmonar intersticial difusa) presentaban mayor rigidez arterial.

In vitro susceptibility to pentavalent antimony in Leishmania infantum strains is not modified during in vitro or in vivo passages but is modified after host treatment with meglumine antimoniate

Background: Leishmaniasis is a common parasitic disease in Southern Europe, caused by Leishmania infantum. The failures of current treatment with pentavalent antimonials are partially attributable to the emergence of antimony-resistant Leishmania strains. This study analyses the in vitro susceptibility to pentavalent antimony of intracellular amastigotes from a range of L. infantum strains, derived from the same infected animal, during in vitro and in vivo passages and after host treatment with meglumine antimoniate. Results: SbV-IC50 values for strains from two distinct isolates from the same host and one stock after two years of culture in NNN medium and posterior passage to hamster were similar (5.0 ± 0.2; 4.9 ± 0.2 and 4.4 ± 0.1 mgSbV/L, respectively). In contrast, a significant difference (P < 0.01, t test) was observed between the mean SbV-IC50 values in the stocks obtained before and after treatment of hosts with meglumine antimoniate (4.7 ± 0.4 mgSbV/L vs. 7.7 ± 1.5 mgSbV/L). Drug-resistance after drug pressure in experimentally infected dogs increased over repeated drug administration (6.4 ± 0.5 mgSbV/L after first treatment vs. 8.6 ± 1.4 mgSbV/L after the second) (P < 0.01, t test). Conclusions: These results confirm previous observations on strains from Leishmania/HIV co-infected patients and indicate the effect of the increasing use of antimony derivatives for treatment of canine leishmaniasis in endemic areas on the emergence of Leishmania antimony-resistant strains.

Rat insulin turnover in vivo

Zucker lean and obese rats were injected under pentobarbital anesthesia with 125I-labeled insulin; at timed intervals from 30 to 120 sec, blood samples were extracted and used for the estimation of insulin levels by RIA. A group of rats from each series was maintained under a constant infusion of noradrenaline. For each insulin determination, a duplicate blood sample containing the same amount of insulin as that used in the RIA, but without the radioactive label, was used as a blank for insulin measurement. The radioactivity in these tubes was then used for the measurement of insulin label per ml blood. From plasma label decay curves and insulin concentrations, the insulin pool size, half-life, and rate of degradation were calculated. Obese rats had higher insulin levels (2.43 nM) and showed less effect of noradrenaline than their lean counterparts, in which insulin distribution volume shrank with noradrenaline treatment. The half-life of plasma insulin was similar in all groups (range, 226-314 sec). Pool size and overall degradation rates were higher in obese (198 femtokatals) than in lean rats (28 femtokatals). It is postulated that obese rats synthesize and cleave much more insulin than lean controls despite their higher circulating levels of insulin.

Nitrogen metabolism in Zucker rats is affected by moderate reduction, but not by moderate incerase in dietary protein

Marked changes in the content of protein in the diet affects the rat"s pattern of growth, but there is not any data on the effects to moderate changes. Here we used a genetically obese rat strain (Zucker) to examine the metabolic modifications induced to moderate changes in the content of protein of diets, doubling (high-protein (HP): 30%) or halving (low-protein (LP): 8%) the content of protein of reference diet (RD: 16%). Nitrogen, energy balances, and amino acid levels were determined in lean (L) and obese (O) animals after 30 days on each diet. Lean HP (LHP) animals showed higher energy efficiency and amino acid catabolism but maintained similar amino acid accrual rates to the lean RD (LRD) group. Conversely, the lean LP (LLP) group showed a lower growth rate, which was compensated by a relative increase in fat mass. Furthermore, these animals showed greater efficiency accruing amino acids. Obesity increased amino acid catabolism as a result of massive amino acid intake; however, obese rats maintained protein accretion rates, which, in the OHP group, implied a normalization of energy efficiency. Nonetheless, the obese OLP group showed the same protein accretion pattern as in lean animals (LLP). In the base of our data, concluded that the Zucker rats accommodate their metabolism to support moderates increases in the content of protein in the diet, but do not adjust in the same way to a 50% decrease in content of protein, as shown by an index of growth reduced, both in lean and obese rats.

Spanish adaptation of the Expectancy Questionnaire (EQ) about alcohol effects in adolescents

Background: Alcohol-related expectancies are especially relevant in relation to alcohol consumption during adolescence. The main aim of this study was to adapt and translate into Spanish (Castilian) the Expectancy Questionnaire (EQ), and to study its psychometric properties in adolescents. Method: The sample was composed of 514 adolescents (57.20% female, mean age = 15.21; SD = .63) who completed the EQ and the alcohol consumption questionnaire AIS-UJI. Results: Confi rmatory factor analysis indicated that an eight-factor model, grouped into two general factors of positive and negative expectancies, had acceptable fi t indices. Cronbach’s alphas ranged from .75 to .96. Finally, the structural equation model showed that positive expectancies were positively related to alcohol use, whereas negative expectancies were negatively related to drinking. Conclusions: Results showed that the Spanish version of the EQ for adolescents is a valid and reliable questionnaire to measure expectancies about alcohol effects.

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.

Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse

Allylnitrile, cis-crotononitrile, and 3,3 -iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5±0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds

Dual role of LldR in regulation of the lldPRD operon, involved in l-Lactate metabolism in Escherichia coli.

The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions 105 to 89) and O2 (positions 22 to 38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.