RESUMEN
The primary response of proliferating bovine pulmonary artery endothelial cells (BPAECs) after X-ray irradiation [≤10 gray (Gy)] is shown to be transient cell-cycle arrest. Accompanying oxidant-linked functional changes within the mitochondria are readily measured, but increased autophagy is not. Radiation-induced apoptosis is negligible in this line-important because cells undergoing apoptosis release oxygen-derived species that can overwhelm/mask the radiation-associated species and their effects that we wish to investigate. Cells irradiated and cultured at 3% oxygen exhibited delayed cell-cycle arrest (6-8 hours after 10 Gy irradiation) compared with those maintained at 20% oxygen (2-4 hours after 10 Gy irradiation). At 3% oxygen, either only during or only after irradiation, results intermediate between 20% and 3% oxygen throughout were obtained. No variability in cell-cycle distribution was observed for unirradiated cells cultured under different prevailing oxygen levels. Mitochondrially localized manganese superoxide dismutase delayed the X-ray-induced cell-cycle changes when over-expressed in BPAEC, indicating superoxide to be one of the key oxygen-derived cytotoxic species involved in the radiobiological response. Also, the peroxynitrite biomarker 3-nitrotyrosine was elevated, whereas hydrogen peroxide levels were not. Lastly, the utility of the BPAEC for screening potential countermeasures to ionizing radiation is demonstrated with some quinoline derivatives. Three of the five compounds appeared mitigative, and all were protective. It is suggested that the oxidation-reduction chemistry of these compounds probably offers a reasonable explanation for their observed ameliorative properties. Furthermore, the results suggest a promising new direction in the search for lead compounds as countermeasures to the effects of ionizing radiation. SIGNIFICANCE STATEMENT: The primary radiological response of proliferating bovine pulmonary artery endothelial cells is cell-cycle arrest, starting soon after X-ray irradiation (1-10 Gy) at 20% O2 but delayed by 4 hours at systemic (3%) O2. Oxygen/superoxide is found to be radio-sensitizing in at least two distinct time windows, during and after the irradiation, with both responses antagonized by various hydroxyquinoline derivatives. Similar responses in many other cell lines are likely to be masked by elevated oxidants associated with apoptosis.
Asunto(s)
Células Endoteliales , Oxidantes , Animales , Bovinos , Rayos X , Oxidantes/farmacología , Superóxidos , Apoptosis/efectos de la radiación , OxígenoRESUMEN
OBJECTIVES: To determine the potential impact of the presence of secondary variant histology on the gene expression profiles of muscle-invasive bladder cancer (MIBC) tumors. METHODS: For six tumors, revised samples were collected from urothelial and secondary variant components (cohort A). The commercial cohort (cohort B) consisted of the anonymized gene expression profiles of 173 patients with MIBC. Samples were obtained from the clinical use of the Decipher Bladder test that were available as part of the Decipher GRID prospective registry (NCT02609269). Secondary variant presence in cohort B was abstracted from institutional pathology reports. For the commercial cohort, only the urothelial carcinoma component was profiled. RESULTS: Molecular subtyping of both urothelial and variant components found micropapillary and nested cases were classified as a luminal subtype. Conversely, the sarcomatoid and small cell cases were classified as basal/squamous or neuroendocrine-like, respectively. For cohort B, 50 (29%) of 173 cases had reported secondary variant histology. Cases with squamous variant had basal profiles, small cell cases expressed neuronal markers, and micropapillary cases were classified as luminal. Sarcomatoid tumors had robust epithelial-mesenchymal transition marker expression. CONCLUSIONS: Our data suggest that in MIBC with secondary variant, the urothelial component can demonstrate an expression profile that closely resembles the variant component.
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Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Perfilación de la Expresión Génica , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Anciano , Biomarcadores de Tumor/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
There is presently no antidote available to treat azide poisoning. Here, the Schiff-base compound Co(II)-2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaenyl dibromide (Co(II)N4[11.3.1]) is investigated to determine if it has the capability to antagonize azide toxicity through a decorporation mechanism. The stopped-flow kinetics of azide binding to Co(II)N4[11.3.1] in the absence of oxygen exhibited three experimentally observable phases: I (fast); II (intermediate); and III (slow). The intermediate phase II accounted for â¼70% of the overall absorbance changes, representing the major process observed, with second-order rate constants of 29 (±4) M-1 s-1 at 25 °C and 70 (±10) M-1 s-1 at 37 °C. The data demonstrated pH independence of the reaction around neutrality, suggesting the unprotonated azide anion to be the attacking species. The binding of azide to Co(II)N4[11.3.1] appears to have a complicated mechanism leading to less than ideal antidotal capability; nonetheless, this cobalt complex does protect against azide intoxication. Administration of Co(II)N4[11.3.1] at 5 min post sodium azide injection (ip) to mice resulted in a substantial decrease of righting-recovery times, 12 (±4) min, compared to controls, 40 (±8) min. In addition, only two out of seven mice "knocked down" when the antidote was administered compared to the controls given toxicant only (100% knockdown).
Asunto(s)
Antídotos/farmacología , Antídotos/uso terapéutico , Cobalto/farmacología , Complejos de Coordinación/farmacología , Bases de Schiff/farmacología , Azida Sódica/antagonistas & inhibidores , Azida Sódica/envenenamiento , Animales , Antídotos/administración & dosificación , Antídotos/química , Cobalto/administración & dosificación , Cobalto/química , Cobalto/uso terapéutico , Complejos de Coordinación/administración & dosificación , Complejos de Coordinación/química , Complejos de Coordinación/uso terapéutico , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Inyecciones Intraperitoneales , Masculino , Ratones , Bases de Schiff/administración & dosificación , Bases de Schiff/química , Bases de Schiff/uso terapéutico , Azida Sódica/administración & dosificación , Tasa de SupervivenciaRESUMEN
Caterpillars of the greater wax moth, Galleria mellonella, are shown to be a useful invertebrate organism for examining mitochondrial toxicants (inhibitors of electron transport) and testing putative antidotes. Administration of sodium azide, sodium cyanide, or sodium (hydro)sulfide by intra-haemocoel injection (through a proleg) results in a dose-dependent paralysed state in the larvae lasting from <1 to ~40â¯min. The duration of paralysis is easily monitored, because if turned onto their backs, the larvae right themselves onto their prolegs once they are able to move again. The efficacy of putative antidotes to the three toxicants can routinely be assessed by observing shortened periods of paralysis with larvae given toxicant and antidote compared to larvae administered only the same dose of toxicant. The validity of the approach is demonstrated with agents previously shown to be antidotal towards cyanide intoxication in mice; namely, sodium nitrite and CoN4[11.3.1] (cobalt(II/III) 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1(7)2,11,13,15-pentaenyl cation). These same compounds are shown to be antidotal towards all three toxicants in the G. mellonella caterpillars; findings that may prove important in relation to azide and sulfide poisonings, for which there are currently no effective antidotes available. The observation that sodium nitrite ameliorates cyanide toxicity in the larvae is additionally interesting because it unambiguously demonstrates that the antidotal action of nitrites does not require the involvement of methemoglobin, contributing to the resolution of an ongoing controversy.
Asunto(s)
Azidas/toxicidad , Cianuros/toxicidad , Complejo IV de Transporte de Electrones/metabolismo , Mariposas Nocturnas/enzimología , Sulfuros/toxicidad , Animales , Complejo IV de Transporte de Electrones/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Larva/efectos de los fármacos , Larva/enzimología , Mariposas Nocturnas/efectos de los fármacosRESUMEN
Four cobalt-containing macrocyclic compounds previously shown to ameliorate cyanide toxicity have been comparatively evaluated with an acute sublethal toxicity model in conscious (unanesthetized) adult male Swiss-Webster mice. All of the compounds (the cobalt-corrins cobalamin and cobinamide, a cobalt-porphyrin, plus a cobalt-Schiff base macrocycle) given 5 min prior to the toxicant dose significantly decreased the righting-recovery time of cyanide-intoxicated mice, but the doses required for maximal antidotal effect varied. Additionally, all of the compounds tested significantly reduced the righting-recovery time when administered at either 1 or 2 min after cyanide intoxication, but none of the compounds tested significantly reduced the righting-recovery time when delivered 5 min after the toxicant dose. Using the lowest effective dose of each compound determined during the first (prophylactic) set of experiments, neuromuscular recovery following cyanide intoxication in the presence/absence of the cobalt-based antidotes was assessed by RotaRod testing. All the compounds tested accelerated recovery of neuromuscular coordination, and no persistent impairment in any group, including those animals that received toxicant and no antidote, was apparent up to 2 weeks postexposures. The relative effectiveness of the cobalt compounds as cyanide antidotes are discussed and rationalized on the basis of the cyanide-binding stoichiometries and stability constants of the Co(III) cyano adducts, together with consideration of the rate constants for axial ligand substitutions by cyanide in the Co(II) forms.
Asunto(s)
Antídotos/farmacología , Cobalto/farmacología , Cianuros/antagonistas & inhibidores , Depuradores de Radicales Libres/toxicidad , Compuestos Organometálicos/farmacología , Animales , Antídotos/química , Cobalto/química , Cianuros/toxicidad , Masculino , Ratones , Estructura Molecular , Compuestos Organometálicos/químicaRESUMEN
Bovine pulmonary artery endothelial cells (BPAEC) respond in a dose-dependent manner to millimolar (0-10) levels of sodium sulfide (NaHS). No measurable increase in caspase-3 activity and no change in the extent of autophagy (or mitophagy) were observed in BPAEC. However, lactate dehydrogenase levels increased in the BPAEC exposed NaHS, which indicated necrotic cell death. In the case of galactose-conditioned BPAEC, the toxicity of NaHS was increased by 30% compared to that observed in BPAEC maintained in the regular glucose-containing culture medium, which indicated a link between mitochondrial oxidative phosphorylation and the mechanism of toxicant action. This is consistent with the widely held view that cytochrome c oxidase (complex IV of the mitochondrial electron-transport system) is the principal molecular target involved in the acute toxicity of "sulfide" (H2S/HS-). In support of this view, elevated NO (which can reverse cytochrome c oxidase inhibition) ameliorated the toxicity of NaHS and, conversely, suppression of endogenous NO production exacerbated the observed toxicity. Respirometric measurements showed the BPAEC to possess a robust sulfide oxidizing system, which was able to out-compete cytochrome c oxidase for available H2S/HS- at micromolar concentrations. This detoxification system has previously been reported by other groups in several cell types, but notably, not neurons. The findings appear to provide some insight into the question of why human survivors of H2S inhalation frequently present at the clinic with respiratory insufficiency/pulmonary edema, while acutely poisoned laboratory animals tend to either succumb to cardiopulmonary paralysis or fully recover without any intervention.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Óxido Nítrico/metabolismo , Arteria Pulmonar/efectos de los fármacos , Sulfuros/toxicidad , Animales , Bovinos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Infusiones Parenterales , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Relación Estructura-Actividad , Sulfuros/administración & dosificaciónRESUMEN
Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxoâhydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis.
Asunto(s)
Biocatálisis , Enzimas/metabolismo , Compuestos Epoxi/química , Hierro , Alquenos/química , Aspergillus nidulans/enzimología , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Cinética , Oxígeno/químicaRESUMEN
The complex of cobalt(II) with the ligand 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaene (CoN4[11.3.1]) has been shown to bind two molecules of cyanide in a cooperative fashion with an association constant of 2.7 (±0.2) × 10(5). In vivo, irrespective of whether it is initially administered as the Co(II) or Co(III) cation, EPR spectroscopic measurements on blood samples show that at physiological levels of reductant (principally ascorbate) CoN4[11.3.1] becomes quantitatively reduced to the Co(II) form. However, following addition of sodium cyanide, a dicyano Co(III) species is formed, both in blood and in buffered aqueous solution at neutral pH. In keeping with other cobalt-containing cyanide-scavenging macrocycles like cobinamide and cobalt(III) meso-tetra(4-N-methylpyridyl)porphine, we found that CoN4[11.3.1] exhibits rapid oxygen turnover in the presence of the physiological reductant ascorbate. This behavior could potentially render CoN4[11.3.1] cytotoxic and/or interfere with evaluations of the antidotal capability of the complex toward cyanide through respirometric measurements, particularly since cyanide rapidly inhibits this process, adding further complexity. A sublethal mouse model was used to assess the effectiveness of CoN4[11.3.1] as a potential cyanide antidote. The administration of CoN4[11.3.1] prophylactically to sodium cyanide-intoxicated mice resulted in the time required for the surviving animals to recover from "knockdown" (unconsciousness) being significantly decreased (3 ± 2 min) compared to that of the controls (22 ± 5 min). All observations are consistent with the demonstrated antidotal activity of CoN4[11.3.1] operating through a cyanide-scavenging mechanism, which is associated with a Co(II) â Co(III) oxidation of the cation. To test for postintoxication neuromuscular sequelae, the ability of mice to remain in position on a rotating cylinder (RotaRod test) was assessed during and after recovery. While intoxicated animals given CoN4[11.3.1] did recover â¼30 min more quickly than controls given only toxicant, there were no indications of longer-term problems in either group, as determined by continuing the RotaRod testing up to 24 h after the intoxications and routine behavioral observations for a further week.
Asunto(s)
Antídotos/farmacología , Cobalto/farmacología , Corrinoides/economía , Corrinoides/farmacología , Cianuros/antagonistas & inhibidores , Compuestos Macrocíclicos/economía , Compuestos Macrocíclicos/farmacología , Animales , Antídotos/química , Antídotos/economía , Cobalto/química , Cobalto/economía , Corrinoides/química , Cianuros/química , Cianuros/toxicidad , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Masculino , Ratones , Bases de Schiff/síntesis química , Bases de Schiff/química , Bases de Schiff/economía , Bases de Schiff/farmacologíaRESUMEN
The Co(III)-containing water-soluble metalloporphyrin cobalt(III) meso-tetra(4-N-methylpyridyl)porphine pentaiodide (Co(III)TMPyP) is a potential cyanide-scavenging agent. The rate of reduction of Co(III)TMPyP by ascorbate is facile enough that conversion to the Co(II)-containing Co(II)TMPyP should occur within minutes at prevailing in vivo levels of the reductant. It follows that any cyanide-decorporating capability of the metalloporphyrin should depend more on the cyanide-binding characteristics of Co(II)TMPyP than those of the administered form, Co(III)TMPyP. Addition of cyanide to buffered aqueous solutions of Co(II)TMPyP (pH 7.4, 25-37 °C) results in quite rapid (k2 = â¼10(3) M(-1) s(-1)) binding/substitution of cyanide anion in the two available axial positions with high affinity (K'ß = 10(10) to 10(11)). Electron paramagnetic resonance spectroscopic measurements and cyclic voltammetry indicate that cyanide induces oxidation to the Co(III)-containing dicyano species. The constraints that these observations put on plausible mechanisms for the reaction of Co(II)TMPyP with cyanide are discussed. Experiments in which Co(III)TMPyP and cyanide were added to freshly drawn mouse blood showed the same sequence of reactions (metalloporphyrin reduction â cyanide binding/substitution â reoxidation) to occur. Therefore, in cyanide-scavenging applications with this metalloporphyrin, we should be taking advantage of both the improved rate of ligand substitution at Co(II) compared to that at Co(III) and the increased affinity of Co(III) for anionic ligands compared to that of Co(II). Finally, using an established sublethal mouse model for cyanide intoxication, Co(III)TMPyP, administered either 5 min before (prophylaxis) or 1 min after the toxicant, is shown to have very significant antidotal capability. Possible explanations for the results of a previous contradictory study, which failed to find any prophylactic effect of Co(III)TMPyP toward cyanide intoxication, are considered.
Asunto(s)
Ácido Ascórbico/química , Cobalto/química , Cianuros/química , Depuradores de Radicales Libres/química , Metaloporfirinas/química , Compuestos Organometálicos/química , Animales , Sitios de Unión , Cobalto/sangre , Técnicas Electroquímicas , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Masculino , Metaloporfirinas/sangre , Ratones , Compuestos Organometálicos/sangre , Oxidación-ReducciónRESUMEN
UNLABELLED: ATP binding cassette transporter A1 (encoded by ABCA1) regulates cholesterol efflux from cells to apolipoproteins A-I and E (ApoA-I and APOE; encoded by APOA1 and APOE, respectively) and the generation of high density lipoproteins. In Abca1 knockout mice (Abca1(ko)), high density lipoproteins and ApoA-I are virtually lacking, and total APOE and APOE-containing lipoproteins in brain substantially decreased. As the ε4 allele of APOE is the major genetic risk factor for late-onset Alzheimer's disease, ABCA1 role as a modifier of APOE lipidation is of significance for this disease. Reportedly, Abca1 deficiency in mice expressing human APP accelerates amyloid deposition and behaviour deficits. We used APP/PS1dE9 mice crossed to Apoe and Apoa1 knockout mice to generate Apoe/Apoa1 double-knockout mice. We hypothesized that Apoe/Apoa1 double-knockout mice would mimic the phenotype of APP/Abca1(ko) mice in regards to amyloid plaques and cognitive deficits. Amyloid pathology, peripheral lipoprotein metabolism, cognitive deficits and dendritic morphology of Apoe/Apoa1 double-knockout mice were compared to APP/Abca1(ko), APP/PS1dE9, and single Apoa1 and Apoe knockouts. Contrary to our prediction, the results demonstrate that double deletion of Apoe and Apoa1 ameliorated the amyloid pathology, including amyloid plaques and soluble amyloid. In double knockout mice we show that (125)I-amyloid-ß microinjected into the central nervous system cleared at a rate twice faster compared to Abca1 knockout mice. We tested the effect of Apoe, Apoa1 or Abca1 deficiency on spreading of exogenous amyloid-ß seeds injected into the brain of young pre-depositing APP mice. The results show that lack of Abca1 augments dissemination of exogenous amyloid significantly more than the lack of Apoe. In the periphery, Apoe/Apoa1 double-knockout mice exhibited substantial atherosclerosis and very high levels of low density lipoproteins compared to APP/PS1dE9 and APP/Abca1(ko). Plasma level of amyloid-ß42 measured at several time points for each mouse was significantly higher in Apoe/Apoa1 double-knockout then in APP/Abca1(ko) mice. This result demonstrates that mice with the lowest level of plasma lipoproteins, APP/Abca1(ko), have the lowest level of peripheral amyloid-ß. Unexpectedly, and independent of amyloid pathology, the deletion of both apolipoproteins worsened behaviour deficits of double knockout mice and their performance was undistinguishable from those of Abca1 knockout mice. Finally we observed that the dendritic complexity in the CA1 region of hippocampus but not in CA2 is significantly impaired by Apoe/Apoa1 double deletion as well as by lack of ABCA1. IN CONCLUSION: (i) plasma lipoproteins may affect amyloid-ß clearance from the brain by the 'peripheral sink' mechanism; and (ii) deficiency of brain APOE-containing lipoproteins is of significance for dendritic complexity and cognition.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Apolipoproteína A-I/deficiencia , Apolipoproteínas E/deficiencia , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/psicología , Eliminación de Gen , Placa Amiloide/genética , Transportador 1 de Casete de Unión a ATP/genética , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacocinética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteínas E/genética , Encéfalo/metabolismo , Encéfalo/patología , Trastornos del Conocimiento/patología , Femenino , Hipocampo/metabolismo , Lipoproteínas/sangre , Masculino , Ratones , Ratones Noqueados , Microinyecciones , Neuritas/patología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Placa Amiloide/patología , Placa Amiloide/psicologíaRESUMEN
Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that bexarotene-liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. To further validate the significance of RXR for these functions, we used mouse embryonic stem (ES) cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene. In vitro data from ES cells confirmed that bexarotene-activated RXR affected neuronal development at different levels, including proliferation of neural progenitors and neuronal differentiation, and stimulated neurite outgrowth. This effect was validated in vivo by demonstrating an increased number of neuronal progenitors after bexarotene treatment in the dentate gyrus of APOE3 and APOE4 mice. In primary neurons, bexarotene enhanced the dendritic complexity characterized by increased branching, intersections, and bifurcations. This effect was confirmed by in vivo studies demonstrating that bexarotene significantly improved the compromised dendritic structure in the hippocampus of APOE4 mice. We conclude that bexarotene-activated RXRs promote genetic programs involved in the neurogenesis and development of neuronal projections and these results have significance for the improvement of cognitive deficits. SIGNIFICANCE STATEMENT: Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. The significance of RXR for these functions was validated in mouse embryonic stem cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene.
Asunto(s)
Diferenciación Celular/fisiología , Dendritas/metabolismo , Neurogénesis/fisiología , Receptores X Retinoide/metabolismo , Tetrahidronaftalenos/farmacología , Animales , Bexaroteno , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Dendritas/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurogénesis/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores X Retinoide/agonistasRESUMEN
There are currently no FDA-approved antidotes for H2S/sulfide intoxication. Sodium nitrite, if given prophylactically to Swiss Webster mice, was shown to be highly protective against the acute toxic effects of sodium hydrosulfide (â¼LD40 dose) with both agents administered by intraperitoneal injections. However, sodium nitrite administered after the toxicant dose did not detectably ameliorate sulfide toxicity in this fast-delivery, single-shot experimental paradigm. Nitrite anion was shown to rapidly produce NO in the bloodstream, as judged by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin, together amounting to less than 5% of the total hemoglobin present. Sulfide-intoxicated mice were neither helped by the supplemental administration of 100% oxygen nor were there any detrimental effects. Compared to cyanide-intoxicated mice, animals surviving sulfide intoxication exhibited very short knockdown times (if any) and full recovery was extremely fast (â¼15 min) irrespective of whether sodium nitrite was administered. Behavioral experiments testing the ability of mice to maintain balance on a rotating cylinder showed no motor impairment up to 24 h post sulfide exposure. It is argued that antagonism of sulfide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity of nitrite rather than formation of methemoglobin.
Asunto(s)
Antídotos/farmacología , Metahemoglobinemia/patología , Nitrito de Sodio/farmacología , Sulfuros/toxicidad , Animales , Aniones/química , Bovinos , Línea Celular , Cianuros/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Inyecciones Intraperitoneales , Masculino , Metahemoglobina/química , Metahemoglobina/metabolismo , Metahemoglobinemia/metabolismo , Ratones , Actividad Motora/efectos de los fármacos , Músculo Esquelético/fisiología , Miocardio/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico/metabolismoRESUMEN
Early growth response gene 1 (Egr1) is a member of the immediate early gene (IEG) family of transcription factors and plays a role in memory formation. To identify EGR1 target genes in brain of Alzheimer's disease (AD) model mice - APP23, we applied chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq). Functional annotation of genes associated with EGR1 binding revealed a set of related networks including synaptic vesicle transport, clathrin-mediated endocytosis (CME), intracellular membrane fusion and transmission of signals elicited by Ca(2+) influx. EGR1 binding is associated with significant enrichment of activating chromatin marks and appears enriched near genes that are up-regulated in the brains of APP23 mice. Among the putative EGR1 targets identified and validated in this study are genes related to synaptic plasticity and transport of proteins, such as Arc, Grin1, Syn2, Vamp2 and Stx6, and genes implicated in AD such as Picalm, Psen2 and App. We also demonstrate a potential regulatory link between EGR1 and its newly identified targets in vivo, since conditions that up-regulate Egr1 levels in brain, such as a spatial memory test, also lead to increased expression of the targets. On the other hand, protein levels of EGR1 and ARC, SYN2, STX6 and PICALM are significantly lower in the brain of adult APP mice than in age-matched wild type animals. The results of this study suggest that EGR1 regulates the expression of genes involved in CME, vesicular transport and synaptic transmission that may be critical for AD pathogenesis.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Redes Reguladoras de Genes/genética , Genoma , Degeneración Nerviosa/genética , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Células COS , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Endocitosis/genética , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/etiología , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/genética , Transducción de Señal/genética , TransfecciónRESUMEN
Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) demonstrated in a mouse model for Alzheimer's disease (AD) that treatment of APP/PS1ΔE9 mice with bexarotene decreased Aß pathology and ameliorated memory deficits. We confirm the reversal of memory deficits in APP/PS1ΔE9 mice expressing human APOE3 or APOE4 to the levels of their nontransgenic controls and the significant decrease of interstitial fluid Aß, but not the effects on amyloid deposition.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Tetrahidronaftalenos/farmacología , Tetrahidronaftalenos/uso terapéutico , Animales , MasculinoRESUMEN
Chronic exposure to arsenic in drinking water, especially in utero or perinatal exposure, can initiate neurological and cognitive dysfunction, as well as memory impairment. Several epidemiological studies have demonstrated cognitive and learning deficits in children with early exposure to low to moderate levels of arsenic, but pathogenic mechanisms or etiology for these deficits are poorly understood. Since in vivo studies show a role for histone acetylation in cognitive performance and memory formation, we examined if prenatal exposure to arsenic causes changes in the epigenomic landscape. We exposed C57Bl6/J mice to 100 µg/L arsenic in the drinking water starting 1 week before conception till birth and applied chromatin immunoprecipitation followed by high-throughput massive parallel sequencing (ChIP-seq) to evaluate H3K9 acetylation pattern in the offspring of exposed and control mice. Arsenic exposure during embryonic life caused global hypo-acetylation at H3K9 and changes in functional annotation with highly significant representation of Krüppel associated box (KRAB) transcription factors in brain samples from exposed pups. We also found that arsenic exposure of adult mice impaired spatial and episodic memory, as well as fear conditioning performance. This is the first study to demonstrate: a) genome wide changes in H3K9 acetylation pattern in an offspring prenatally exposed to arsenic, and b) a connection between moderate arsenic exposure and cognitive impairment in adult mice. The results also emphasize the applicability of Next Generation Sequencing methodology in studies aiming to reveal the role of environmental factors, other than dietary restriction, in developmental reprogramming through histone modifications during embryonic development.
Asunto(s)
Arsénico/toxicidad , Histonas/genética , Histonas/metabolismo , Trastornos de la Memoria/inducido químicamente , Efectos Tardíos de la Exposición Prenatal , Acetilación/efectos de los fármacos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Disfunción Cognitiva/etiología , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Agua Potable , Femenino , Estudio de Asociación del Genoma Completo , Masculino , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Embarazo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
ATP-binding cassette transporter A1 (ABCA1) transporter regulates cholesterol efflux and is an essential mediator of high-density lipoprotein (HDL) formation. In amyloid precursor protein (APP) transgenic mice, Abca1 deficiency increased amyloid deposition in the brain paralleled by decreased levels of Apolipoprotein E (ApoE). The APOEε4 allele is the major genetic risk factor of sporadic Alzheimer's disease (AD). Here, we reveal the effect of Abca1 deficiency on phenotype in mice expressing human ApoE3 or ApoE4. We used APP/E3 and APP/E4 mice generated by crossing APP/PS1ΔE9 transgenic mice to human APOE3- and APOE4-targeted replacement mice and examined Abca1 gene dose effect on amyloid deposition and cognition. The results from two behavior tests demonstrate that lack of one copy of Abca1 significantly exacerbates memory deficits in APP/E4/Abca1(-/+) but not in APP/E3/Abca1(-/+) mice. The data for amyloid plaques and insoluble amyloid-ß (Aß) also show that Abca1 hemizygosity increases Aß deposition only in APP/E4/Abca1(-/+) but not in APP/E3/Abca1(-/+) mice. Our in vivo microdialysis assays indicate that Abca1 deficiency significantly decreases Aß clearance in ApoE4-expressing mice, while the effect of Abca1 on Aß clearance in ApoE3-expressing mice was insignificant. In addition, we demonstrate that plasma HDL and Aß42 levels in APP/E4/Abca1(-/+) mice are significantly decreased, and there is a negative correlation between plasma HDL and amyloid plaques in brain, suggesting that plasma lipoproteins may be involved in Aß clearance. Overall, our results prove that the presence of functional Abca1 significantly influences the phenotype of APP mice expressing human ApoE4 and further substantiate therapeutic approaches in AD based on ABCA1-APOE regulatory axis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/deficiencia , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Transportador 1 de Casete de Unión a ATP , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Encéfalo/metabolismo , Encéfalo/patología , Colesterol/metabolismo , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Condicionamiento Psicológico/fisiología , Ensayo de Inmunoadsorción Enzimática , Miedo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microdiálisis , Mutación/genética , Fragmentos de Péptidos/metabolismo , Fenotipo , Presenilina-1/genéticaRESUMEN
A hallmark of Alzheimer disease (AD) is the deposition of amyloid ß (Aß) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aß(40) aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aß(42) and decreases Aß(42) toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I(KO) mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I(KO) mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aß oligomer levels, Aß plaque load, or levels of insoluble Aß in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aß isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I(KO) mice, insoluble Aß(40) is increased more than 10-fold, and Aß(42) is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I(KO) mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aß toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Apolipoproteína A-I/fisiología , Encéfalo/patología , Angiopatía Amiloide Cerebral/patología , Trastornos de la Memoria/patología , Presenilina-1/fisiología , Animales , Conducta Animal , Western Blotting , Encéfalo/metabolismo , Células Cultivadas , Angiopatía Amiloide Cerebral/etiología , Colesterol/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas para Inmunoenzimas , Aprendizaje por Laberinto , Trastornos de la Memoria/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de SecuenciaRESUMEN
The liver X receptors (LXRalpha and LXRbeta) are transcription factors that control the expression of genes primarily involved in cholesterol metabolism. In the brain, in addition to normal neuronal function, cholesterol metabolism is important for APP proteolytic cleavage, secretase activities, Abeta aggregation and clearance. Particularly significant in this respect is LXR mediated transcriptional control of APOE, which is the only proven risk factor for late onset Alzheimer's disease. Using a transactivation reporter assay for screening pharmacologically active compounds and off patent drugs we identified the proton pump inhibitor Lansoprazole as an LXR agonist. In secondary screens and counter-screening assays, it was confirmed that Lansoprazole directly activates LXR, increases the expression of LXR target genes in brain-derived human cell lines, and increases Abca1 and Apo-E protein levels in primary astrocytes derived from wild type but not LXRalpha/beta double knockout mice. Other PPIs activate LXR as well, but the efficiency of activation depends on their structural similarities to Lansoprazole. The identification of a widely used drug with LXR agonist-like activity opens the possibility for systematic preclinical testing in at least two diseases--Alzheimer's disease and atherosclerosis.