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1.
Mol Biol Rep ; 48(5): 4527-4535, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-34145535

RESUMEN

The mechanism by which reactive oxygen species (ROS) produced by oxidative stress promote cellular senescence has been studied in detail. This study aimed to verify the preventive or therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-Ex) on the production of ROS induced by oxidative stress in human skin fibroblasts and clarify the mechanisms that promote cellular senescence. In a system where H2O2 was applied to skin fibroblasts, we assessed the effects of the application of MSC-Ex before and after oxidative stress and measured the fluctuations in several signaling molecules involved in subsequent intracellular stress responses. Exosomes were isolated from MSCs (MSC-Ex) and normal human dermal fibroblasts (NHDFs, NHDF-Ex) before and after exposure to H2O2. NHDFs were treated with exosomes before and after exposure to H2O2. mRNA expression (aquaporin-1 and aquaporin-3) and hyaluronan secretion associated with skin moisturization were reduced by H2O2 treatment, whereas MSC-Ex reversed these effects. The cellular senescence induced by H2O2 was also reproduced in fibroblasts. Specifically, the downregulation of SIRT1 led to increased acetylated p53 expression over time, which induced the expression of p21, a downstream molecule of p53, and arrested the cell cycle, leading to cell senescence. MSC-Ex enhanced these signal transduction systems. MSC-Ex was also effective at blocking the increase of ß-galactosidase activity and accumulation of ROS in cells. This effect was stronger than that of NHDF-Ex. MSC-Ex were found to act defensively against epidermal and cellular senescence induced by oxidative stress.


Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Exosomas/genética , Estrés Oxidativo/genética , Sirtuina 1/genética , Proteína p53 Supresora de Tumor/genética , Acuaporina 1/genética , Acuaporina 3/genética , Senescencia Celular/genética , Exosomas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Piel/efectos de los fármacos , Piel/crecimiento & desarrollo , Piel/metabolismo
2.
Archaea ; 2016: 8734894, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27799846

RESUMEN

The oxidation of guanine (G) to 7,8-dihydro-8-oxoguanine (GO) forms one of the major DNA lesions generated by reactive oxygen species (ROS). The GO can be corrected by GO DNA glycosylases (Ogg), enzymes involved in base excision repair (BER). Unrepaired GO induces mismatched base pairing with adenine (A); as a result, the mismatch causes a point mutation, from G paired with cytosine (C) to thymine (T) paired with adenine (A), during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeon Thermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315) shares significant sequence homology with the DNA glycosylases of Methanocaldococcus jannaschii (MjaOgg) and Sulfolobus solfataricus (SsoOgg). The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed in Escherichia coli and purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP) lyase activities on GO/N (where N is A, T, G, or C) mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG) can. These results indicate that TvoOgg-encoding TVG_RS00315 is a member of the Ogg2 family of T. volcanium.


Asunto(s)
ADN Glicosilasas/metabolismo , ADN/metabolismo , Guanina/análogos & derivados , Thermoplasma/enzimología , ADN Glicosilasas/química , ADN Glicosilasas/genética , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Guanina/metabolismo , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Temperatura , Thermoplasma/genética
3.
Biol Pharm Bull ; 37(12): 1982-5, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25451847

RESUMEN

The bursa of Fabricius (BF) is a unique primary lymphoid organ, and among vertebrates is unique to birds. Despite its importance to the immune systems of various avian species, little is known of the molecular mechanisms underlying early BF development. In the present study, we demonstrated that apoptosis occurs during early development of the bursa of Fabricius in chicken embryos. Initial histological analyses of BF morphogenesis in chicken embryos led to the hypothesis that formation of the bursal lumen correlates with fusion of vacuoles, which appear in the cloacal epithelial bud. Using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) analysis and immunostaining with an anti-cleaved (activated) caspase-3 antibody, we detected multiple apoptotic cells around these vacuoles. In further experiments, treatments with a caspase inhibitor caused abnormal bursal lumen in vivo. The present data indicate that apoptosis may play important roles in BF morphogenesis in chickens.


Asunto(s)
Apoptosis/fisiología , Bolsa de Fabricio/embriología , Embrión de Pollo/citología , Embrión de Pollo/crecimiento & desarrollo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Bolsa de Fabricio/citología , Inhibidores de Caspasas/farmacología
4.
Biol Pharm Bull ; 37(3): 481-5, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24583867

RESUMEN

The genome of the facultative anaerobic thermoacidophilic archaeon Thermoplasma volcanium contains the open-reading frames (ORFs) tvsod and tvogg, which are predicted to encode a putative superoxide dismutase and an 8-oxoguanine DNA glycosylase, respectively. Tvsod is immediately upstream of tvogg, and these two ORFs are aligned in a head-to-tail manner in a single operon. A previous study showed that T. volcanium contains an ORF (TVN0292) encoding the ferric uptake regulator (Fur) and that the T. volcanium Fur protein (TvFur) binds to its own promoter in a metal-dependent manner in vitro. Here, we demonstrated that TvFur also binds to the tvsod-tvogg promoter and determined the TvFur-binding sequences in the tvsod-tvogg promoter by DNaseI footprinting analysis. These results suggest that Fur is required for resistance against reactive oxygen species in this facultative anaerobic archaeon.


Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Operón , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Thermoplasma/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Represoras/metabolismo , Thermoplasma/metabolismo
5.
Biochem Biophys Res Commun ; 441(1): 25-30, 2013 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-24125720

RESUMEN

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.


Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Aprendizaje , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Cromosomas de los Mamíferos/genética , Cruzamientos Genéticos , Femenino , Estudios de Asociación Genética , Proteínas Fluorescentes Verdes/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Péptidos/metabolismo , Plásmidos/metabolismo , Canales de Potasio/química , Sitios de Carácter Cuantitativo/genética
6.
PLoS One ; 16(3): e0248027, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33662018

RESUMEN

Molecular chaperon SERPINA3 colocalizes with accumulated amyloid peptide in Alzheimer's disease (AD) patient's brain. From the QTL analysis, we narrowed down Serpina3 with two SNPs in senescence-accelerated mouse prone (SAMP) 8 strain. Our study showed SAMP8 type Serpina3 prolonged retention of oligomeric Aß 42 for longer duration (72 hr) while observing under transmission electron microscope (TEM). From Western blot results, we confirmed presence of Aß 42 oligomeric forms (trimers, tetramers) were maintained for longer duration only in the presences of SAMP8 type Serpina3. Using SH-SY5Y neuroblastoma cell line, we observed until 36 hr preincubated Aß 42 with SAMP8 type Serpina3 caused neuronal cell death compared to 12 hr preincubated Aß 42 with SAMR1 or JF1 type Serpina3 proteins. Similar results were found by extending this study to analyze the effect of polymorphism of SERPINA3 gene of the Japanese SNP database for geriatric research (JG-SNP). We observed that polymorphic SERPINA3 I308T (rs142398813) prolonged toxic oligomeric Aß 42 forms till 48 hr in comparison to the presence wild type SERPINA3 protein, resulting neuronal cell death. From this study, we first clarified pathogenic regulatory role of polymorphic SERPINA3 in neurodegeneration.


Asunto(s)
Proteínas de Fase Aguda/genética , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Polimorfismo de Nucleótido Simple , Serpinas/genética , Proteínas de Fase Aguda/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/análisis , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Humanos , Masculino , Ratones , Fragmentos de Péptidos/análisis , Multimerización de Proteína , Sitios de Carácter Cuantitativo , Serpinas/metabolismo
7.
Neurochem Res ; 34(4): 660-9, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19247832

RESUMEN

The SAMP8 strain spontaneously develops learning and memory deficits with characteristics of aging, and is a good model for studying the mechanism of cognitive dysfunction with age. Oxidative stress occurs systemically in SAMP8 from early on in life and increases with aging. Neuropathological changes such as the deposition of A beta, hyperphosphorylation of tau, impaired development of dendritic spines, and sponge formation, and neurochemical changes were found in the SAMP8 brain. These changes may be partially mediated by oxidative stress. Oxidative damage is a major factor in neurodegenerative disorders and aging. A decline in the respiratory control ratio suggesting mitochondrial dysfunction was found in the brain of SAMP8. The rise in oxidative stress following mitochondrial dysfunction may trigger neuropathological and neurochemical changes, disrupting the development of neural networks in the brain in SAMP8.


Asunto(s)
Envejecimiento , Herencia , Modelos Animales , Enfermedades Neurodegenerativas , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Espinas Dendríticas/patología , Ratones , Ratones Endogámicos , Mitocondrias/fisiología , Red Nerviosa/patología , Red Nerviosa/fisiología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo , Fosforilación , Proteínas tau/metabolismo
8.
Brain Res ; 1141: 214-7, 2007 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-17303091

RESUMEN

Senescence-accelerated mouse P8 (SAMP8) mice show deficits of learning and memory at an early age. However, no evidence of neurochemical changes was found in the hippocampus of SAMP8 at an early age. After electric shock in the passive avoidance test, SAMR1 (normal aging mice) showed biphasic responses in the phosphorylated CREB (p-CREB) level in the hippocampal CA1 region: an early peak detected at 1 to 3 h was followed by a marked drop at 6 h, and a second peak rise starting after 9 to 12 h after electric stimulation. On the other hand, SAMP8 manifested one peak in the p-CREB level 9 h after the stimulation. Since the phosphorylation of CREB plays an important role for synaptic plasticity and consolidation of long-term memory, the impairment of CREB phosphorylation in the hippocampal CA1 region of SAMP8 may cause learning and memory deficits.


Asunto(s)
Envejecimiento/patología , Envejecimiento/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hipocampo/metabolismo , Animales , Reacción de Prevención/fisiología , Conducta Animal , Electrochoque/métodos , Regulación de la Expresión Génica/fisiología , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Ratones Endogámicos , Fosforilación , Factores de Tiempo
9.
Biomed Pharmacother ; 61(7): 427-34, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17644302

RESUMEN

Oligonol is produced from the oligomerization of polyphenols (typically proanthocyanidin from a variety of fruits such as lychees, grapes, apples, persimmons, etc.) and contains catechin-type monomers and oligomers of proanthocyanidins. The ability of Oligonol to affect infection-dependent eye inflammation, locomotion and longevity in senescence-accelerated prone mice (SAMP8) (a model of senescence acceleration and geriatric disorders with increased oxidative stress and neuronal deficit) was investigated. Oligonol (60mg/kg) significantly modulated the extent of inflammation scores in the eye of SAMP8 mice. Examination of the mice indicated infection with mouse hepatitis virus and pinworm (Syphacia obvelata) in both males and females and with the intestinal protozoa (trichomonad) in males. A comparison of the two groups (using log-rank test) and the difference in the mean life span between groups (using Student's t-test) indicated significant differences in survival (p=0.043) and the mean life span (p=0.033) in male SAMP8 mice. Oligonol increased the mean life span and this was statistically significant. In the open-field locomotive test, the 7-week-old SAMP8 mice crossed more than 40 partitioned lines in 1min. At 48-week-old control untreated male SAMP8 crossed 2 lines. The Oligonol-treated 48-week-old male SAMP8 mice crossed 17 lines however. The improved locomotive activity was statistically significant even after 36weeks in the Oligonol-treated male SAMP8 but this was not the case throughout the time course of the study in the Oligonol-treated female SAMP8. Thus Oligonol treatment to SAMP8 mice modulated the severity of infection-dependent inflammation, prolonged life-span and significantly improved locomotive activity indicating potential benefit to aging-associated diseases such as Alzheimer's or Parkinson's diseases. This presents potential for further research to define infection-dependent inflammation associated with degenerative conditions and the molecular mechanism of dietary antioxidant protection.


Asunto(s)
Catequina/análogos & derivados , Inflamación/tratamiento farmacológico , Enfermedades Parasitarias en Animales/complicaciones , Fenoles/uso terapéutico , Virosis/complicaciones , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Conducta Animal , Catequina/farmacología , Catequina/uso terapéutico , Suplementos Dietéticos , Modelos Animales de Enfermedad , Ojo/efectos de los fármacos , Ojo/patología , Femenino , Inflamación/etiología , Inflamación/fisiopatología , Longevidad/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Actividad Motora/efectos de los fármacos , Enfermedades Parasitarias en Animales/tratamiento farmacológico , Enfermedad de Parkinson/tratamiento farmacológico , Fenoles/farmacología , Tasa de Supervivencia , Factores de Tiempo , Virosis/tratamiento farmacológico
10.
Biofactors ; 26(2): 135-46, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16823099

RESUMEN

The antioxidative activity and ameliorative effects on memory impairment by sulfur-containing compounds which occur in Allium vegetables such as onion and garlic were investigated. The antioxidative activities of S-alk(en)yl-L-cysteines and their sulfoxides, volatile alk(en)yl disulfides and trisulfides, and vinyldithiins were examined by using human low-density lipoprotein. It was elucidated that the alk(en)yl substituents and the number of sulfur atoms in the compounds were important for the antioxidative activities. To demonstrate the ameliorative effects on memory impairment, onion extract and synthesized di-n-propyl trisulfide were administered to senescence-accelerated mouse P8. The behavioral experiments showed that onion extract and di-n-propyl trisulfide had highly ameliorative effect of memory impairment. Furthermore, it was found that the hippocampus lipid hydroperoxide in senescence-accelerated mouse P8 was decreased by the administration of di-n-propyl trisulfide. These results suggest that di-n-propyl trisulfide contained in onion ameliorates memory impairment in SAMP8 mouse by its antioxidant effect.


Asunto(s)
Allium/química , Antioxidantes/farmacología , Cisteína/análogos & derivados , Cisteína/farmacología , Memoria/efectos de los fármacos , Sulfuros/farmacología , Envejecimiento/fisiología , Animales , Disulfuros/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Peróxidos Lipídicos/biosíntesis , Lipoproteínas LDL/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Cebollas/química , Fosfatidilcolinas/metabolismo , Extractos Vegetales/farmacología
11.
Forensic Sci Int ; 265: 182-5, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26986505

RESUMEN

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) procedure for the simultaneous determination of nine local anesthetic drugs (procaine, mepivacaine, lidocaine, ropivacaine, oxybuprocaine, tetracaine, bupivacaine, T-caine and dibucaine) in human serum is described. The chromatographic separation was performed on a Mightysil-RP-18 GP II column (2.0mm×150mm, particle size 5µm). The mobile phase consisted of 10mM acetic ammonium buffer (pH 5.4) and acetonitrile and was delivered at a flow rate of 0.20mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the nine local anesthetic drugs added to the human serum was performed with an Oasis(®) HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 10-100ng/mL. The recoveries of these drugs were in the range of 81.4-144%. The standard deviation (SD) values for all analytes were <0.10 for both intraday and interday accuracy and precision. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic applications. The sensitive and selective method offers the opportunity for the simultaneous screening and quantification, for clinical and forensic purposes, of almost all local anesthetics available in Japan.


Asunto(s)
Anestésicos Locales/sangre , Trastornos Relacionados con Sustancias/diagnóstico , Cromatografía Liquida , Toxicología Forense , Humanos , Valor Predictivo de las Pruebas , Detección de Abuso de Sustancias/métodos , Trastornos Relacionados con Sustancias/sangre , Espectrometría de Masas en Tándem
12.
Physiol Behav ; 84(4): 505-10, 2005 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-15811384

RESUMEN

Genetic analysis of learning and memory deficits (LMD) in senescence-accelerated mouse P8 (SAMP8) was performed by cross-mating SAMP8 and Japanese Fancy Mouse 1 (JF1). The incidence of LMD in the F2 generation showed a 3:1 segregation ratio of mice with LMD to normal mice, and the incidence of LMD in the backcross generation of the F1 to JF1 parental strain was in agreement with a 1:1 ratio of mice with LMD to normal mice. Estimation of the number of genes involved in the development of LMD using Wright's formula showed that at least two to four genes are involved. These results suggest that the inheritance of LMD is polygenically controlled and that there may be a single major gene, but this locus is not sex-linked. Moreover, hormonal influence on the development of LMD in SAMP8 females is of a genotype-dependent manner.


Asunto(s)
Envejecimiento/genética , Reacción de Prevención/fisiología , Discapacidades para el Aprendizaje/genética , Trastornos de la Memoria/genética , Retención en Psicología/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Genotipo , Hormonas Esteroides Gonadales/fisiología , Hibridación Genética , Endogamia , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Herencia Multifactorial/genética , Índice de Severidad de la Enfermedad , Factores Sexuales
13.
Arch Gerontol Geriatr ; 57(3): 417-22, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23806789

RESUMEN

SAMP8 exhibits accelerated aging and a short lifespan. Insulin-like growth factor-1 receptor (IGF-1R)/FOXO pathway is associated with aging. Phosphorylation of IGF-1R, Akt, and FOXO1 was found to be increased during aging in the liver of SAMR1 normal aging mice. However, significant decreases in the phosphorylation of IGF-1R and Akt were observed in the liver of SAMP8 during aging compared with that in SAMR1, whereas phosphorylation of FOXO1 was markedly increased with age in SAMP8. In addition, the protein level of FOXO1 was decreased with age in SAMP8. Protein phosphatase 2A (PP2A) directly dephosphorylates FOXO1. Significant reduction of PP2A activity was observed in the liver nucleus of SAMP8. These results suggest the possibility that the increased FOXO1 phosphorylation might occur by the decreased activity of PP2A, resulting in the decrease in the protein level of FOXO1 in SAMP8. Furthermore, FOXO1 regulates longevity and the expression of antioxidant enzymes such as Mn-SOD and catalase. The expression of Mn-SOD and catalase was significantly decreased in the liver of SAMP8. Therefore, it is possible that the elevation of phosphorylated FOXO1 level with age causes a short lifespan in SAMP8.


Asunto(s)
Factores de Transcripción Forkhead/análisis , Hígado/química , Envejecimiento/fisiología , Animales , Western Blotting , Proteína Forkhead Box O1 , Hígado/enzimología , Hígado/fisiología , Masculino , Ratones , Ratones Mutantes , Proteína Oncogénica v-akt/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína Fosfatasa 2/metabolismo , Receptor IGF Tipo 1/metabolismo , Superóxido Dismutasa/metabolismo
14.
Forensic Sci Int ; 227(1-3): 100-2, 2013 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-23273940

RESUMEN

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique was developed for the simultaneous determination of five non-steroidal anti-inflammatory oxicam drugs (ampiroxicam, tenoxicam, piroxicam, meloxicam and lornoxicam) in human plasma. These five oxicam drugs and isoxicam (internal standard) were extracted from human plasma with an Oasis(®) MAX cartridge column and analysed on a Unison UK-C18 column (2.0 mm × 100 mm, 3 µm) with an acetonitrile:10mM formic ammonium buffer (pH 3.0) (50:50) mobile phase at 0.20 ml/min at 37°C. The analytes were detected using a tandem mass spectrometer, equipped with an electrospray ion source (ESI). The instrument was used in multiple-reaction-monitoring (MRM) mode. The extraction yields from a 200 µl human plasma sample (containing 10 ng of each drugs) with the Oasis(®) MAX cartridge column were 93.3-102.5%. The detection limits were 0.01-6.5 ng/ml (S/N=3). Our developed method is very useful for the simultaneous determination of five oxicam (non-steroidal anti-inflammatory) drugs in human plasma by LC/MS/MS.


Asunto(s)
Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/química , Cromatografía Liquida/métodos , Toxicología Forense/métodos , Humanos , Meloxicam , Estructura Molecular , Piroxicam/análogos & derivados , Piroxicam/sangre , Piroxicam/química , Espectrometría de Masas en Tándem/métodos , Tiazinas/sangre , Tiazinas/química , Tiazoles/sangre , Tiazoles/química
15.
Arch Gerontol Geriatr ; 54(2): e1-7, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-21784539

RESUMEN

SAMP8 mice show spontaneously accelerated aging and a short life span with systemic accumulation of oxidative stress. Nrf2 translocates into the nucleus upon oxidative stress and induces the expression of detoxifying and antioxidant enzymes. Recently, several studies reported that Nrf2 is associated with aging and various diseases. In the present study, we investigated the levels of Nrf2 nuclear translocation and phosphorylation of Akt and GSK-3ß in livers of SAMP8 and normal aging SAMR1 mice. The protein level of Nrf2 in the nucleus of the liver was significantly decreased in SAMP8 at 10 months old compared with that in age-matched SAMR1. The protein level of Keap1, which anchors Nrf2 in the cytoplasm, did not differ between SAMP8 and SAMR1. In addition, the mRNA expression of Nrf2 in the liver of SAMP8 was significantly lower than that of SAMR1. Moreover, mRNA levels of detoxification and antioxidant enzymes, GSTa1 and NQO1, were significantly decreased in SAMP8 compared with SAMR1. These results indicate that a higher level of oxidative stress in SAMP8 might be caused by a lower level of Nrf2. Furthermore, the phosphorylation of Akt and GSK-3ß was significantly decreased in the liver of SAMP8 at 10 months old. Recent studies have suggested that the Akt/GSK-3ß signaling pathway is involved in the nuclear translocation of Nrf2. Therefore, it is suggested that the reduction of the translocation of Nrf2 into the nucleus might be induced by a decrease of GSK-3ß phosphorylation, resulting in an increase of oxidative stress in SAMP8 mice.


Asunto(s)
Envejecimiento/fisiología , Glucógeno Sintasa Quinasa 3/fisiología , Factor 2 Relacionado con NF-E2/fisiología , Envejecimiento/metabolismo , Animales , Núcleo Celular/química , Núcleo Celular/fisiología , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos , Factor 2 Relacionado con NF-E2/análisis , Factor 2 Relacionado con NF-E2/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteína Oncogénica v-akt/fisiología , Estrés Oxidativo/fisiología , Fosforilación , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/fisiología
16.
J Gen Appl Microbiol ; 58(6): 465-73, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23337582

RESUMEN

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.


Asunto(s)
Proteínas Arqueales/metabolismo , Cationes Bivalentes/metabolismo , ADN de Archaea/metabolismo , Regulación de la Expresión Génica Arqueal , Hierro/metabolismo , Regiones Promotoras Genéticas/genética , Thermoplasma/metabolismo , Factores de Transcripción/metabolismo , Proteínas Arqueales/genética , Clonación Molecular , Huella de ADN , ADN de Archaea/genética , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Thermoplasma/clasificación , Thermoplasma/genética , Factores de Transcripción/genética
17.
Biol Pharm Bull ; 28(4): 615-8, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15802797

RESUMEN

The senescence-accelerated mouse (SAM) is known as a murine model for accelerated aging. The SAMP8 shows age-related deficits of learning and memory at an earlier age than control mice (SAMR1). We investigated the changes in oligodendrocytes in the brain of SAMP8, using immunohistochemistry for myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) as an oligodendrocyte marker. SAMP8 at 10 months old showed a decrease in MBP-immunoreactivity (IR) and CNP-IR in the hippocampal CA1 subfield, compared with SAMR1. There were no significant differences in MBP and CNP old in the cerebral cortex and the optic tract between SAMR1 and SAMP8 at 10 months. Furthermore, we measured the area of MBP-IR in the CA1 subfield of both strains and found that the area of MBP-IR in SAMP8 had decreased progressively with age, compared with SAMR1. These results suggest that age-related degeneration of oligodendrocytes had occurred in the hippocampus of SAMP8.


Asunto(s)
Envejecimiento/patología , Hipocampo/patología , Oligodendroglía/patología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Envejecimiento/metabolismo , Animales , Hipocampo/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos , Proteína Básica de Mielina/metabolismo , Oligodendroglía/metabolismo
18.
Glycoconj J ; 20(6): 375-84, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15238702

RESUMEN

We have sought an endogenous membrane bound sialidase acting at neutral pH in immune system, because the removal of sialic acid from cell surfaces will affect the cell-cell interaction directly or indirectly. The levels of activity of unique membrane-bound sialidase at neutral pH and also soluble sialidase are high in the thymus but low in the spleen and lymph nodes. These are thought to be plasma membrane and cytosolic types based on the behavior of inhibition by Cu(2+) and 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid. Newly synthesized 5-bromo-4-chloro-3-indolyl-N-acetylnueraminic acid was used for histochemical staining of sialidase-positive thymic cells, and the results showed positive cells sparsely distributed in the corticomedullar region or medullary region of the thymus. They expressed immunoglobulin and Mac-1 antigen on their surfaces. These cells must therefore be of a B cell lineage, not a T cell lineage. We also found that some vessels in the thymus were sialidase-positive.


Asunto(s)
Inmunoglobulinas/metabolismo , Antígeno de Macrófago-1/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidasa/biosíntesis , Timo/metabolismo , Animales , Linfocitos B/metabolismo , Comunicación Celular , Linaje de la Célula , Membrana Celular/metabolismo , Cobre/química , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Ratones , Ratones Endogámicos C3H , Ácido N-Acetilneuramínico/farmacología , Neuraminidasa/metabolismo , Fosfatos/metabolismo , Bazo/metabolismo , Linfocitos T/metabolismo , Factores de Tiempo
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