RESUMEN
Expression quantitative trait loci (eQTL) studies have functionalized nucleic acid variants through the regulation of gene expression. Although most eQTL studies only examine the effects of single variants on transcription, a more complex process of variant-variant interaction (epistasis) may regulate transcription. Herein, we describe a tool called interaction QTL (iQTL) designed to efficiently detect epistatic interactions that regulate gene expression. To maximize biological relevance and minimize the computational and hypothesis testing burden, iQTL restricts interactions such that one variant is within a user-defined proximity of the transcript (cis-regulatory). We apply iQTL to a data set of 183 smallpox vaccine study participants with genome-wide association study and gene expression data from unstimulated samples and samples stimulated by inactivated vaccinia virus. While computing only 0.15% of possible interactions, we identify 11 probe sets whose expression is regulated through a variant-variant interaction. We highlight the functional epistatic interactions among apoptosis-related genes, DIABLO, TRAPPC4 and FADD, in the context of smallpox vaccination. We also use an integrative network approach to characterize these iQTL interactions in a posterior network of known prior functional interactions. iQTL is an efficient, open-source tool to analyze variant interactions in eQTL studies, providing better understanding of the function of epistasis in immune response and other complex phenotypes.
Asunto(s)
Apoptosis/genética , Epistasis Genética , Sitios de Carácter Cuantitativo , Viruela/genética , Programas Informáticos , Adolescente , Adulto , Proteínas Reguladoras de la Apoptosis , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Redes Reguladoras de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Viruela/inmunología , Vacuna contra Viruela/inmunología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
OBJECTIVE: Diabetes is a known risk factor for mortality in Coronavirus disease 2019 (COVID-19) patients. Our objective was to identify prevalence of hyperglycaemia in COVID-19 patients with and without prior diabetes and quantify its association with COVID-19 disease course. RESEARCH DESIGN AND METHODS: This observational cohort study included all consecutive COVID-19 patients admitted to John H Stroger Jr. Hospital, Chicago, IL from March 15, 2020 to May 3, 2020 and followed till May 15, 2020. The primary outcome was hospital mortality, and the studied predictor was hyperglycaemia [any blood glucose ≥7.78 mmol/L (140 mg/dL) during hospitalization]. RESULTS: Of the 403 COVID-19 patients studied, 51 (12.7%) died; 335 (83.1%) were discharged while 17 (4%) were still in hospital. Hyperglycaemia occurred in 228 (56.6%) patients; 83 of these hyperglycaemic patients (36.4%) had no prior history of diabetes. Compared to the reference group no-diabetes/no-hyperglycaemia patients the no-diabetes/hyperglycaemia patients showed higher mortality [1.8% versus 20.5%, adjusted odds ratio 21.94 (95% confidence interval 4.04-119.0), P < 0.001]; improved prediction of death (P = 0.01) and faster progression to death (P < 0.01). Hyperglycaemia within the first 24 and 48 h was also significantly associated with mortality (odds ratio 2.15 and 3.31, respectively). CONCLUSIONS: Hyperglycaemia without prior diabetes was common (20.6% of hospitalized COVID-19 patients) and was associated with an increased risk of and faster progression to death. Development of hyperglycaemia in COVID-19 patients who do not have diabetes is an early indicator of progressive disease.
Asunto(s)
Glucemia/análisis , COVID-19/mortalidad , Hiperglucemia/mortalidad , Adulto , Anciano , COVID-19/sangre , Femenino , Mortalidad Hospitalaria , Hospitalización , Humanos , Hiperglucemia/sangre , Masculino , Persona de Mediana EdadRESUMEN
Regular consumers of caffeine had higher muscle tension after three or more hours of abstinence than low caffeine consumers. This difference was absent after double-blind administration of caffeine citrate or placebo. In a discriminative reaction time test, caffeine treatment improved performance. Among subjects receiving placebo, anxiety was highly correlated with prior caffeine use, suggesting that even a brief abstinence may produce anxiety in the regular user.
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Ansiedad/etiología , Cafeína/farmacología , Contracción Muscular/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/fisiopatología , Adulto , Humanos , Síndrome de Abstinencia a Sustancias/complicacionesRESUMEN
MOTIVATION: The development of genome-wide capabilities for genotyping has led to the practical problem of identifying the minimum subset of genetic variants relevant to the classification of a phenotype. This challenge is especially difficult in the presence of attribute interactions, noise and small sample size. METHODS: Analogous to the physical mechanism of evaporation, we introduce an evaporative cooling (EC) feature selection algorithm that seeks to obtain a subset of attributes with the optimum information temperature (i.e. the least noise). EC uses an attribute quality measure analogous to thermodynamic free energy that combines Relief-F and mutual information to evaporate (i.e. remove) noise features, leaving behind a subset of attributes that contain DNA sequence variations associated with a given phenotype. RESULTS: EC is able to identify functional sequence variations that involve interactions (epistasis) between other sequence variations that influence their association with the phenotype. This ability is demonstrated on simulated genotypic data with attribute interactions and on real genotypic data from individuals who experienced adverse events following smallpox vaccination. The EC formalism allows us to combine information entropy, energy and temperature into a single information free energy attribute quality measure that balances interaction and main effects. AVAILABILITY: Open source software, written in Java, is freely available upon request.
Asunto(s)
Mapeo Cromosómico/métodos , Análisis Mutacional de ADN/métodos , Bases de Datos Genéticas , Evolución Molecular , Genotipo , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Simulación por Computador , Modelos Genéticos , Modelos Estadísticos , Datos de Secuencia MolecularRESUMEN
BACKGROUND: The recurrence rate for paraesophageal hernias (PEH) can be as high as 30% following laparoscopic repair. The aim of this study was to determine the severity of symptoms in patients with recurrences and the need for reoperation 10 years after surgery. METHODS AND PROCEDURES: Consecutive laparoscopic paraesophageal cases performed at a single institution between 1993 and 1996 were identified from the institution's foregut database. Patients were asked about the presence and severity of symptoms (heartburn, chest pain, regurgitation, and dysphagia). Patients were also asked whether they had (1) been diagnosed with hernia recurrence or (2) undergone repeat surgical intervention. RESULTS: Complete follow-up was obtainable in 31 of the total of 52 patients (60%). The proportion of patients reporting moderate/severe symptoms was less at 10 years than preoperatively: heartburn 12% versus 54% (p < 0.001), chest pain 9% versus 36% (p = 0.01), regurgitation 6% versus 50% (p < 0.001), and dysphagia 3% versus 30% (p = 0.001). Two patients underwent repeat surgical intervention for symptomatic recurrences within the first postoperative year. Eight more patients have been diagnosed with hernia recurrences on either contrast esophagram or upper endoscopy but had not required reoperation. At ten years, more patients with hernia recurrence had heartburn than those who did not have recurrences (60% versus 14%; p < 0.05). CONCLUSIONS: Despite a hiatal hernia recurrence rate of 32% 10 years after surgery, laparoscopic PEH was a successful procedure in the majority of patients; most remained symptomatically improved and required no further intervention 10 years after surgery.
Asunto(s)
Enfermedades del Esófago/cirugía , Hernia Hiatal/cirugía , Laparoscopía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Recurrencia , Estadísticas no ParamétricasRESUMEN
BACKGROUND: ReliefF is a nearest-neighbor based feature selection algorithm that efficiently detects variants that are important due to statistical interactions or epistasis. For categorical predictors, like genotypes, the standard metric used in ReliefF has been a simple (binary) mismatch difference. In this study, we develop new metrics of varying complexity that incorporate allele sharing, adjustment for allele frequency heterogeneity via the genetic relationship matrix (GRM), and physicochemical differences of variants via a new transition/transversion encoding. METHODS: We introduce a new two-dimensional transition/transversion genotype encoding for ReliefF, and we implement three ReliefF attribute metrics: 1.) genotype mismatch (GM), which is the ReliefF standard, 2.) allele mismatch (AM), which accounts for heterozygous differences and has not been used previously in ReliefF, and 3.) the new transition/transversion metric. We incorporate these attribute metrics into the ReliefF nearest neighbor calculation with a Manhattan metric, and we introduce GRM as a new ReliefF nearest-neighbor metric to adjust for allele frequency heterogeneity. RESULTS: We apply ReliefF with each metric to a GWAS of major depressive disorder and compare the detection of genes in pathways implicated in depression, including Axon Guidance, Neuronal System, and G Protein-Coupled Receptor Signaling. We also compare with detection by Random Forest and Lasso as well as random/null selection to assess pathway size bias. CONCLUSIONS: Our results suggest that using more genetically motivated encodings, such as transition/transversion, and metrics that adjust for allele frequency heterogeneity, such as GRM, lead to ReliefF attribute scores with improved pathway enrichment.
RESUMEN
Brain reperfusion causes prompt, severe, and prolonged protein synthesis suppression and increased phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha(P)] in hippocampal CA1 and hilar neurons. The authors hypothesized that eIF2alpha(P) dephosphorylation would lead to recovery of protein synthesis. Here the effects of insulin, which activates phosphatases, were examined by immunostaining for eIF2alpha(P) and autoradiography of in vivo 35S amino acid incorporation. Rats resuscitated from a 10-minute cardiac arrest were given 0, 2, 10 or 20 U/kg of intravenous insulin, underwent reperfusion for 90 minutes, and were perfusion fixed. Thirty minutes before perfusion fixation, control and resuscitated animals received 500 microCi/kg of 35S methionine/cysteine. Alternate 30-microm brain sections were autoradiographed or immunostained for eIF2alpha(P). Controls had abundant protein synthesis and no eIF2alpha(P) in hippocampal neurons. Untreated reperfused neurons in the CA1, hilus, and dentate gyrus had intense staining for eIF2alpha(P) and reduced protein synthesis; there was little improvement with treatment with 2 or 10 U/kg of insulin. However, with 20 U/kg of insulin, these neurons recovered protein synthesis and were free of eIF2alpha(P). These results show that the suppression of protein synthesis in the reperfused brain is reversible; they support a causal association between eIF2alpha(P) and inhibition of protein synthesis, and suggest a mechanism for the neuroprotective effects of insulin.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Hipocampo/metabolismo , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Ataque Isquémico Transitorio/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Animales , Autorradiografía , Hipocampo/irrigación sanguínea , Hipocampo/patología , Ataque Isquémico Transitorio/patología , Masculino , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Ratas , Ratas Long-EvansRESUMEN
Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.
Asunto(s)
Encéfalo/metabolismo , Calpaína/metabolismo , Ataque Isquémico Transitorio/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Animales , Encéfalo/efectos de los fármacos , Calcio/farmacología , Proteínas de Unión al Calcio/farmacología , Calpaína/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Factor 4G Eucariótico de Iniciación , Femenino , Cinética , Masculino , Conejos , Ratas , Reperfusión , Fracciones Subcelulares/metabolismoRESUMEN
Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha), we used an antibody specific to phosphorylated eIF-2 alpha [eIF-2(alpha P)] to study the regional and cellular distribution of eIF-2(alpha P) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that approximately 1% of all eIF-2 alpha is phosphorylated in controls, eIF-2(alpha P) is not reduced by up to 30 minutes of ischemia, and eIF-2(alpha P) is increased approximately 20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(alpha P) to astrocytes in normal brains, a massive increase in eIF-2(alpha P) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(alpha P) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(alpha P) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(alpha P) inhibits translation initiation, make a compelling case that eIF-2(alpha P) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.
Asunto(s)
Isquemia Encefálica/metabolismo , Daño por Reperfusión/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Inmunohistoquímica , Masculino , Fosforilación , Ratas , eIF-2 Quinasa/análisisRESUMEN
Iron-mediated peroxidation of brain lipids is known to occur during reperfusion following cardiac arrest. Since in vitro damage to DNA is caused by similar iron-dependent peroxidation, we tested whether free radical damage to genomic DNA also develops during reperfusion following cardiac arrest and resuscitation. Genomic DNA was isolated from the cerebral cortex in (i) normal dogs, (ii) dogs subjected to a 20-min cardiac arrest, and (iii) dogs resuscitated from a 20-min cardiac arrest and then allowed to reperfuse for 2 or 8 h. DNA strand nicks were evaluated by in vitro labeling of newly created 3' and 5' termini. DNA base damage was evaluated utilizing reaction with piperidine prior to labeling of 5' termini. The 3' DNA termini were labeled before and after digestion with exonuclease III, and the 5' DNA termini were labeled before and after treatment with piperidine. In vitro experiments with genomic DNA damaged by oxygen radicals verified that these labeling methods identified radical damage. In the experimental animal groups, terminal incorporation and electrophoretic mobility of brain nuclear DNA are not significantly changed either by 20 min of complete brain ischemia or during the first 8 h of reperfusion. We conclude that genomic DNA is not extensively damaged during cardiac arrest and early reperfusion, and therefore such DNA damage does not appear to be an important early aspect of the neurologic injury that accompanies cardiac arrest and resuscitation.
Asunto(s)
Encéfalo/metabolismo , Daño del ADN , ADN/metabolismo , Paro Cardíaco/metabolismo , Hierro/farmacología , Daño por Reperfusión/metabolismo , Animales , Corteza Cerebral/química , Perros , Exodesoxirribonucleasas/metabolismo , Radicales Libres , Hidróxidos , Radical Hidroxilo , PlásmidosRESUMEN
Unilateral intrastriatal microinjections of alpha-methyl-p-tyrosine followed by systemic amphetamine treatment produced turning ipsilateral to the side of the injection in rats from 12 days of age to maturity, but not in rats younger than 12 days. These results are consistent with other findings that indicate rapid development of the striatal dopaminergic system and dopaminergic component of the amphetamine stereotypy in the second postnatal week.
Asunto(s)
Conducta/efectos de los fármacos , Catecolaminas/metabolismo , Cuerpo Estriado/metabolismo , Dextroanfetamina/farmacología , Conducta Estereotipada/efectos de los fármacos , Envejecimiento , Animales , Cuerpo Estriado/efectos de los fármacos , Interacciones Farmacológicas , Femenino , Lateralidad Funcional , Humanos , Inyecciones , Masculino , Metiltirosinas/administración & dosificación , Metiltirosinas/farmacología , RatasRESUMEN
Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.
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Isquemia Encefálica/metabolismo , Degeneración Nerviosa/metabolismo , Daño por Reperfusión/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/fisiología , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Calpaína/metabolismo , Diferenciación Celular/fisiología , Circulación Cerebrovascular/fisiología , Aminoácidos Excitadores/metabolismo , Radicales Libres/metabolismo , Genes Inmediatos-Precoces/fisiología , Sustancias de Crecimiento/metabolismo , Humanos , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/biosíntesis , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Transducción de Señal/fisiologíaRESUMEN
Free-radical reactions, known to occur in the reperfused brain, damage DNA in vitro. We therefore examined the hypothesis that thymine glycols and thymine dimers, which are known to block transcription and are formed by free radical mechanisms, are formed in brain DNA during reoxygenation following ischemia. Such biochemical lesions could account for the failure of protein synthesis that occurs following an ischemic insult. Large dogs were anesthetized, instrumented, and divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without resuscitation; (3) 20-min cardiac arrest, resuscitation and 2 h reperfusion; and (4) 20-min cardiac arrest, resuscitation and 8 h reperfusion. Genomic DNA was isolated from the cerebral cortex. Thymine glycols were labeled by reduction with [3H]NaBH4. Pyrimidine dimers were determined by ELISA using antibody prepared against ultraviolet irradiated DNA. The data was evaluated by Kruskal-Wallis ANOVA with alpha = 0.05. The rabbit antibodies detected the thymine dimer content in 10 pg UV irradiated DNA but did not react with normal DNA. Borohydride labeling qualitatively detected thymine glycols generated by treatment of DNA with osmium tetroxide. There was no difference between the DNAs from the experimental groups in the content of thymine glycols or pyrimidine dimers (P = 0.608 and P = 0.219, respectively). We conclude that significant quantities of thymine glycols and thymine dimers are not formed in brain DNA during post-ischemic reperfusion. Therefore, the inhibition of brain protein synthesis during reperfusion, observed by other investigators, is unlikely to be caused by interruption of transcription by these species.
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Isquemia Encefálica/fisiopatología , ADN/química , Dímeros de Pirimidina/análisis , Daño por Reperfusión/metabolismo , Timina/análogos & derivados , Animales , Daño del ADN , Perros , Ensayo de Inmunoadsorción Enzimática , Radicales Libres , Timina/análisis , Transcripción GenéticaRESUMEN
1-Ethyl-2-methyl-3-hydroxy-pyrid-4-one (EMHP), a low molecular weight iron chelator that is soluble in hydrocarbon solvents and presumably in lipids, was studied for in vitro inhibition of radical-mediated peroxidation of DNA. We also investigated the acute toxicity of EMHP by administering 40, 100, and 300 mg/kg intravenously to Wistar rats, and we then examined the in vivo effect of the 40 mg/kg dose following a 10-min cardiac arrest and resuscitation in rats. EMHP prevented iron-dependent radical-mediated DNA breaks of the supercoiled plasmid Bluescribe by the Fenton reagent (400 microM iron, 30 microM H2O2) when present at EMHP/Fe ratios of 16:1 and 32:1. The 300-mg/kg dose was lethal in 3 of 5 normal rats, and the 100-mg/kg dose was associated with excessive mortality post-resuscitation. The 40-mg/kg dose was well tolerated post-resuscitation, but it did not improve either 3-day survival or neurologic outcome.
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Paro Cardíaco/tratamiento farmacológico , Quelantes del Hierro/uso terapéutico , Resucitación , Animales , Encéfalo/irrigación sanguínea , ADN/efectos de los fármacos , Radicales Libres , Paro Cardíaco/terapia , Quelantes del Hierro/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas , Daño por Reperfusión/prevención & controlRESUMEN
Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.
Asunto(s)
Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Creatina Quinasa/biosíntesis , Proteínas de Choque Térmico/biosíntesis , ARN Mensajero/biosíntesis , Daño por Reperfusión/metabolismo , Animales , Northern Blotting , Isquemia Encefálica/genética , Creatina Quinasa/genética , Perros , Paro Cardíaco/terapia , Proteínas de Choque Térmico/genética , Isoenzimas , Biosíntesis de Proteínas/fisiología , Daño por Reperfusión/genética , Resucitación , Ribosomas/metabolismo , Transcripción Genética/fisiologíaRESUMEN
These experiments examine the effects of arachidonate with respect to cell death, radical-mediated injury, Ca2+ mobilization, and formation of ser-51-phosphorylated eukaryotic initiation factor 2alpha [eIF2alpha(P)]. It is known that during brain ischemia the concentration of free arachidonate can reach 180 microM, and during reperfusion oxidative metabolism of arachidonate leads to generation of superoxide that can reduce stored ferric iron and promote lipid peroxidation. During early brain reperfusion, we have shown an approximately 20-fold increase in eIF2alpha(P) which maps to vulnerable neurons that display inhibition of protein synthesis. Here in neuronally differentiated NB-104 cells, equivalent cell death (assessed by LDH release) was induced by 40 microM arachidonate and 20 microM cumene hydroperoxide (CumOOH, a known alkoxyl radical generator). In these injury models (1) radical inhibitors (BHA, BHT, and the lipophilic iron chelator EMHP) block CumOOH-induced cell death but do not block arachidonate-induced death; (2) 40 microM arachidonate (but not up to 40 microM CumOOH) rapidly induces Ca2+ release from intracellular stores; (3) both 40 microM arachidonate and 20 microM CumOOH induce intense immunostaining for eIF2alpha(P); and (4) the elF2alpha(P) immunostaining induced by CumOOH but not that induced by arachidonate is completely blocked by anti-radical intervention with EMHP. Arachidonate-induced formation of eIF2alpha(P) and cell death do not require iron-mediated radical mechanisms and are associated with Ca2+ release from intracellular stores; however, radical-mediated injury also induces both eIF2alpha(P) and cell death without release of intracellular Ca2+. Our data link eIF2alpha(P) formation during brain reperfusion to two established injury mechanisms that may operate concurrently.
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Ácido Araquidónico/farmacología , Calcio/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Neuronas/metabolismo , Animales , Derivados del Benceno/farmacología , Muerte Celular , Células Cultivadas , Radicales Libres , Neuronas/citología , Neuronas/efectos de los fármacos , Oxidantes/farmacología , RatasRESUMEN
The effect of the combination of two drugs, i.e. lidoflazine (a calcium antagonist), and deferoxamine (an iron chelator) was evaluated following 15 min global brain ischaemia (GBI) and reperfusion in dogs in a randomized blind study. GBI was produced by complete cardiac arrest of 15 min duration. Histopathological analysis performed on in situ fixed brains 40 h post-resuscitation revealed diffuse microhaemorrhages in the control group. These were noted rarely in the treatment group, the mean value of foci of microhaemorrhages/20 low power fields (LPF) being 5.2 in the treatment group versus 28 in the control group (p less than 0.001). Diffuse coagulative necrosis of neurons (ischaemic cell change) in the cerebral cortex, especially lamina 3, hippocampus, striatum, brain stem and cerebellum was present in all cases. Quantitation of the degree of cellular damage obtained by counting the number of anoxic neurons (in consistent regions of the brain) with the use of an image analysis system, revealed no significant difference between the 2 groups. The mean percentages of the ischaemic neurons in the control group in the various areas studied were: parietal cortex, 22.25; hippocampus, 50.37 and cerebellum (Purkinje cells), 66.75; and in the treatment group 25.3, 55.04 and 70.6 respectively. Thus, the lidoflazine-deferoxamine regimen significantly reduced the incidence of microhaemorrhages in the brain, but it did not have any protective effect against anoxic neuronal injury 40 h post-ischaemia in this experimental model of GBI of 15 min duration.
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Bloqueadores de los Canales de Calcio/uso terapéutico , Deferoxamina/uso terapéutico , Hierro/metabolismo , Ataque Isquémico Transitorio/tratamiento farmacológico , Lidoflazina/uso terapéutico , Piperazinas/uso terapéutico , Animales , Perros , Quimioterapia Combinada , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patologíaRESUMEN
Apoptosis is a process of cell suicide, the mechanisms of which are encoded in the genomes of all higher eukaryotes. The mechanisms involved in apoptosis suggest that the process is based on a viral defense originally developed in primitive multicelled eukaryotes and that the fundamental execution platform of the process involves 1) inhibition of protein synthesis at the level of translation initiation, 2) proteolysis specifically involving degradation of DNA repair mechanisms, and 3) polynucleotide degradation. In mammals this execution platform is regulated by a complex molecular signaling system that includes feedback mechanisms tending toward activation of all elements of the execution platform if only one element is initially engaged. Tissue ischemia and reperfusion activate elements of the apoptosis system, which thus represents a therapeutic target for emerging treatment approaches to preserve cellular integrity in critical organs such as the heart and brain.
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Apoptosis , Animales , Apoptosis/fisiología , Caenorhabditis elegans/embriología , Caspasas , Humanos , Isquemia/patología , Daño por Reperfusión/patología , Transducción de SeñalRESUMEN
Caffeine pretreatment was studied for its effects on d-amphetamine-induced locomotor activity. Caffeine (30 mg/kg) was given either simultaneously with or at one of several intervals (0.5, 1.5, 4.5, 12, and 13.5 hours) before d-amphetamine (1.5 mg/kg). Enhancement of d-amphetamine activity occurred with simultaneous and 30 min caffeine pretreatment. However, when given 12 or 13.5 hours before d-amphetamine, caffeine diminished the activity, measured in stabilimeter cages. Several doses of caffeine (7.5, 15, 30, and 60 mg/kg) were given in multiple treatments ending 12 hours before d-amphetamine (1.5 mg/kg) to determine the effective doses for attenuation of d-amphetamine-induced activity. Only 30 and 60 mg caffeine doses reduced d-amphetamine activity while also interfering with body weight gain.
Asunto(s)
Cafeína/farmacología , Dextroanfetamina/farmacología , Actividad Motora/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Interacciones Farmacológicas , Masculino , RatasRESUMEN
Novel tastes preceded a range of caffeine doses (10-80 mg/kg) in a taste aversion training trial. One week later rats which had doses of 30 mg or higher showed strong aversions as measured by a single bottle consumption test. The 10 and 20 mg dose produced the most hyperactivity and apparently enhanced intake of the taste paired with caffeine. During the training trial, rats receiving the 80 mg dose exhibited copious gapes and chin-rubs, mimetic responses to noxious tastes. Gapes also occurred in these subjects during the aversion test. Consumption was more sensitive than mimetic responding as a measure of the aversive effects of caffeine. Only the 80 mg dose produced neophobia. Tests with isotonic injections indicated that tonicity was not the source of the aversions.