RESUMEN
OBJECTIVE: The aim of this study was to investigate the surface roughness of lithium disilicates (LS2s) polished using various polishing systems. MATERIALS AND METHODS: Two types of LS2 (A, Amber Mill and E, IPS e.max CAD) were polished using LS2-specific polishing systems (L-Edenta, L-Jota), a zirconia-specific polishing system (Z-Jota), and a conventional ceramic polishing system (P-Shofu) (n = 8 per group). The compositions of different polishing systems were analyzed using EDS. Surface roughness was measured using confocal laser scanning microscopy and analyzed using EDS and SEM. ANOVA and Tukey's tests were used for the statistical analyses (p = 0.05). RESULTS: The polishing systems were mainly composed of C, O, and Si. The L-Jota group exhibited rougher surfaces than the other groups. Amber Mill exhibited higher surface roughness than IPS e.max CAD (p⟨0.001). Among the polishing systems, the L-Jota group presented the highest roughness value (pp⟨0.001). The surface roughness of the AL-Jota group was higher than that of the other groups. CONCLUSIONS: A sufficiently smooth surface can be achieved without a LS2-specific polishing system. Further, the same polishing system can have different effects depending on the type of LS2.
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Ámbar , Pulido Dental , Cerámica , Diseño Asistido por Computadora , Porcelana Dental , Ensayo de Materiales , Propiedades de SuperficieRESUMEN
Recent studies have indicated a potential correlation between rheumatoid arthritis (RA) and periodontal inflammation. We undertook this study to verify whether RA mediates periodontitis-like phenotypes in experimental mouse models of RA and to explore the role of nicotinamide phosphoribosyltransferase (NAMPT) in periodontal inflammation during RA pathogenesis. Periodontal inflammation and alveolar bone loss have been reported in mice with collagen-induced arthritis (CIA) and in genetically modified tumor necrosis factor-α (TNF-α) transgenic (TG) mouse models. Among the adipokines examined in our study, NAMPT expression was markedly upregulated in the periodontal ligament (PDL) tissues in RA mouse models and in human PDL cells stimulated by the proinflammatory cytokines, interleukin (IL) 1ß and TNF-α. When NAMPT was overexpressed with the Nampt-synthesizing adenovirus vector (Ad- Nampt), the PDL cells exhibited an increased expression of cytokines (IL6), chemokines (IL8 and chemokine [C-C motif] ligand 5 [CCL5]), inflammatory mediators (cyclooxygenase 2 [COX-2]), and matrix-degrading enzymes (matrix metalloproteinase [MMP] 1 and MMP3). Inhibition of NAMPT by the intracellular NAMPT (iNAMPT) inhibitor, FK866, or by the sirtuin inhibitor, nicotinamide, in PDL cells led to inhibition of the IL1ß or Ad- Nampt-induced upregulation of catabolic factors, whereas treatment with recombinant NAMPT protein or blockade of extracellular NAMPT (eNAMPT) with blocking antibody did not. Moreover, NAMPT inhibition by the intraperitoneal or intragingival injection of FK866 in CIA mice inhibited periodontal tissue damage, under conditions of RA. Thus, our results verified the co-occurrence of RA and periodontal inflammation using experimental mouse models of RA, suggesting that iNAMPT in PDL cells plays a pivotal role in the pathogenesis of RA-mediated periodontal inflammation by regulating the expression levels of catabolic genes, such as IL6, IL8, CCL5, COX-2, MMP1, and MMP3.
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Artritis Reumatoide/metabolismo , Nicotinamida Fosforribosiltransferasa/fisiología , Periodontitis/metabolismo , Animales , Tobillo/diagnóstico por imagen , Artritis Experimental/metabolismo , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inflamación/metabolismo , Masculino , Maxilar/diagnóstico por imagen , Ratones , Ratones Transgénicos , Fenotipo , Reacción en Cadena de la Polimerasa , Microtomografía por Rayos XRESUMEN
Reciprocal interactions between cancer cells and the tumor microenvironment drive multiple clinically significant behaviors including dormancy, invasion, and metastasis as well as therapy resistance. These microenvironment-dependent phenotypes share typical characteristics with cancer stem cells (CSC). However, it is poorly understood how metabolic stress in the confined tumor microenvironment contributes to the emergence and maintenance of CSC-like phenotypes. Here, we demonstrate that chronic metabolic stress (CMS) in a long-term nutrient deprivation induces a Wnt-dependent phenoconversion of non-stem cancer cells toward stem-like state and this is reflected in the transcriptome analysis. Addition of Wnt3a as well as transfection of dominant-negative Tcf4 establishes an obligatory role for the Wnt pathway in the acquisition of CSC-like characteristics in response to metabolic stress. Furthermore, systematic characterization for multiple single cell-derived clones and negative enrichment of CD44+/ESA+ stem-like cancer cells, all of which recapitulate stem-like cancer characteristics, suggest stochastic adaptation rather than selection of pre-existing subclones. Finally, CMS in the tumor microenvironment can drive a CSC-like phenoconversion of non-stem cancer cells through stochastic state transition dependent on the Wnt pathway. These findings contribute to an understanding of the metabolic stress-driven dynamic transition of non-stem cancer cells to a stem-like state in the tumor metabolic microenvironment.
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Neoplasias de la Mama/patología , Células Madre Neoplásicas/citología , Estrés Fisiológico/fisiología , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/patología , Esferoides Celulares/patología , Transcripción Genética/genética , Activación Transcripcional/genética , Células Tumorales Cultivadas , Microambiente Tumoral/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In concentration-QTc modeling, oscillatory functions have been used to characterize biological rhythms in QTc profiles. Fitting such functions is not always feasible because it requires sufficient electrocardiograph sampling. In this study, drug concentration and QTc data were simulated using a published biological QTc model (oscillatory functions). Then, linear mixed-effect models and the biological model were fitted and evaluated in terms of biases, precisions, and qualities of inferences. The simpler linear mixed-effect model with day and time as a factor variables provided similar accuracy of the concentration-QTc slope estimates to the complex biological model and was able to accurately predict the drug-induced QTc prolongation with less than 1 ms bias, despite its empirical nature to account for biological rhythm. The current study may guide a concentration-QTc modeling strategy that can be easily prespecified, does not suffer from poor convergence, and achieves little bias in drug-induced QTc estimates.
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The structural, magnetic and electron-transport properties of Mn(2)Pt(1-x)Co(x)Sn(x = 0, 0.3, 0.5, 0.7, 1) ribbons prepared by arc-melting and melt-spinning were investigated. The rapidly quenched alloys with x = 0 and 0.3 were found to crystallize in the inverse tetragonal structure, but the structure transformed into inverse cubic as x increased to 0.5. At room temperature, the samples are ferro or ferrimagnetic, and the Curie temperature increases by 225 K from 370 K for Mn(2)PtSn (x = 0) to 595 K for Mn(2)CoSn (x = 1). The measured anisotropy constants for the inverse-tetragonal alloys are on the order of 1 Merg cm(-3) at room temperature. The ribbons are moderately conducting with the room temperature resistivities being between 0.4 and 8.4 mΩ cm. Interestingly, the thermal coefficient of resistivity transforms from positive to negative and the magnetoresistance transforms from negative to positive as the value of x reaches 0.5.
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The role of immortal DNA strands that co-segregate during mitosis of asymmetrically self-renewing distributed stem cells (DSCs) is unknown. Previously, investigation of immortal DNA strand function and molecular mechanisms responsible for their nonrandom co-segregation was precluded by difficulty in identifying DSCs and immortal DNA strands. Here, we report the use of two technological innovations, selective DSC expansion and establishment of H2A.Z chromosomal asymmetry as a specific marker of 'immortal chromosomes,' to investigate molecular properties of immortal chromosomes and opposing 'mortal chromosomes' in cultured mouse hair follicle DSCs. Although detection of the respective suppressive and activating H3K27me3 and H3K4me3 epigenetic marks on immortal chromosomes was similar to randomly segregated chromosomes, detection of both was lower on mortal chromosomes destined for lineage-committed sister cells. This global epigenomic feature of nonrandom co-segregation may reveal a mechanism that maintains an epigenome-wide 'poised' transcription state, which preserves DSC identity, while simultaneously activating sister chromosomes for differentiation.
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Cromosomas/metabolismo , ADN/metabolismo , Epigénesis Genética , Histonas/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Linaje de la Célula/genética , Segregación Cromosómica/genética , Cromosomas/química , ADN/genética , Metilación de ADN , Folículo Piloso/citología , Folículo Piloso/metabolismo , Histonas/genética , Ratones , Mitosis , Células Madre/citologíaRESUMEN
The structural, magnetic and electron-transport properties of cubic Mn3Ga have been investigated. The alloys prepared by arc melting and melt-spinning show an antiferromagnetic spin order at room temperature but undergo coupled structural and magnetic phase transitions at 600 and 800 K. First-principles calculations show that the observed magnetic properties are consistent with that of a cubic Mn3Ga crystallizing in the disordered Cu3Au-type structure. The samples exhibit metallic electron transport with a resistance minimum near 30 K, followed by a logarithmic upturn below the minimum. The observed anomaly in the low-temperature resistivity has been discussed as a consequence of electron scattering at the low-lying excitations of the structurally disordered Mn3Ga lattice.
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Galio/química , Campos Magnéticos , Manganeso/química , Modelos Químicos , Modelos Moleculares , Simulación por Computador , Conductividad Eléctrica , Ensayo de Materiales , Conformación Molecular , Transición de FaseRESUMEN
Chronic gastritis is a disease that occurs in one in every 10 persons in Korea. Endoscopic examination is needed to diagnose chronic gastritis in western medicine, but it causes patients pain, long period of examinations and financial burden. In KM (Korean Medicine), on the other hand, it can be known whether stomach is abnormal or not through a pulse diagnosis. The 'Guan' position of the right wrist is related to a stomach in KM. Thus, the pulse wave of the right-hand "Guan" of patients with chronic gastritis and the healthy were measured. Then, the diagnostic parameter and features to distinguish between the patients with chronic gastritis and the healthy were discovered. Through P-H curve, consequently, it can be concluded that the pulse waves of patients with chronic gastritis appear as a floating pulse, whereas the pulse waves of the healthy appear as a normal pulse.
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Gastritis/fisiopatología , Análisis de la Onda del Pulso/instrumentación , Análisis de la Onda del Pulso/métodos , Anciano , Pueblo Asiatico , Enfermedad Crónica , Femenino , Gastritis/diagnóstico , Humanos , Corea (Geográfico) , Masculino , Persona de Mediana Edad , Pulso ArterialRESUMEN
Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.
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Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cartílago/metabolismo , Condrocitos/metabolismo , Osteoartritis/metabolismo , Receptor fas/biosíntesis , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cartílago/patología , Células Cultivadas , Condrocitos/patología , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Transgénicos , Osteoartritis/genética , Osteoartritis/patología , Transducción de Señal/genética , Receptor fas/genéticaRESUMEN
The adulteration of dietary supplements with drugs is potentially dangerous for human health. In this study, a method was used to test simultaneously for the presence of three synthetic PDE-5 inhibitors (sildenafil, vardenafil and tadalafil), and sibutramine and its two major metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) using ultra-performance liquid chromatography (UPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF MS) in dietary supplements. This approach with UPLC/Q-TOF MS uses the high accurate mass of six compounds for identification and has a short run time. The recovery was from 87% to 113%; precision was less than 12.8%. The limit of detection and limit of quantification were from 0.4 to 2.0 µg kg(-1) and from 1.3 to 6.0 µg kg(-1), respectively. This method allows easy and fast analysis and detection of diverse adulterants.
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Carbolinas/análisis , Cromatografía Liquida/métodos , Ciclobutanos/análisis , Suplementos Dietéticos , Espectrometría de Masas/métodos , Ciclobutanos/química , Límite de Detección , Estándares de Referencia , Reproducibilidad de los Resultados , TadalafiloRESUMEN
Billions of neurons are interconnected in the central nervous system (CNS). Identification of specific neuronal circuit is indispensable for understanding the relationship between structure and function in the CNS. The midbrain dopamine (DA) neuron system consists of the retrorubral area (A8), the substantia nigra (SN; A9) and the ventral tegmental area (VTA; A10). We hypothesized that genetic methods using cell-type-specific promoters may offer the possibility to express tracer molecules in DA neurons to facilitate neuronal tracing. To address this, we used the 2.5 kb rat tyrosine hydroxylase (TH) promoter in adenovirus or adeno-associated virus (AAV) to express tracers specifically in DA neurons. We found that stereotaxic injection of TH promoter containing adenoviral construct resulted in cell-type-specific transgene expression in the noradrenaline (NA) neurons of the locus coeruleus (LC). However, it caused a significant toxicity to DA neurons in the SN. In contrast, stereotaxic injection of TH promoter containing AAV to the SN resulted in cell-type-specific transgene expression in DA neurons with no detectable toxicity. Taken together, our results demonstrate that it is possible to selectively trace DA neuronal circuits in rodent brains using the TH promoter in the context of AAV.
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Dopamina/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Mesencéfalo/metabolismo , Neuronas/metabolismo , Tirosina 3-Monooxigenasa/genética , Animales , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Vías Nerviosas/fisiología , Regiones Promotoras Genéticas , Ratas , Transgenes/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Magnetic poly epsilon-caprolactone (PCL) nanoparticles were prepared in a well shaped spherical form by the o/w emulsion method. The influence of some preparative variables on the size and surface property was investigated. Nanoparticles were smooth, well individualized and homogeneous in size. The presence of magnetite and its superparamagnetic characteristic were confirmed by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM), respectively. The anti-cancer drug was encapsulated in the magnetic nanoparticle during preparation. A typical release behavior was observed for 30 days. In vitro experiment of magnetic susceptibility under external magnetic field demonstrated that the magnetic PCL nanoparticles have sufficient magnetic susceptibility for a potential magnetic drug carrier for targeted delivery.
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Portadores de Fármacos , Magnetismo , Nanopartículas , Poliésteres/administración & dosificación , Cisplatino/administración & dosificación , Cisplatino/química , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Poliésteres/química , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , GemcitabinaRESUMEN
Human cathepsin K, a cysteine proteinase of the papain family, has been recognized as a potential drug target for the treatment of osteoporosis. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel antiresorptive drugs. Now, we report the pharmacological properties of OST-4077 [furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide] as a novel selective cathepsin K inhibitor. Human and rat cathepsin K were inhibited in vitro by OST-4077 with the IC50 values of 11 and 427 nM, respectively. OST-4077 suppressed bone resorption induced by rabbit osteoclasts (IC50, 37 nM) but did not affect bone mineralization or cellular alkaline phosphatase activity in MC3T3-E1 cells. Parathyroid hormone-induced bone resorption was inhibited in a dose-dependent manner in thyroparathyroidectomized rats gavaged with a single dose of OST-4077 (ED50, 69 mg/kg). When given orally twice daily for 4 weeks to 3-month-old ovariectomized (OVX) rats, OST-4077 dose-dependently prevented bone loss, as monitored by bone densitometry, ash content, and urinary excretion of deoxypyridinoline. No change in serum osteocalcin in the OVX rats by OST-4077 suggested that bone formation might not be affected by the agent. In summary, OST-4077 selectively inhibited bone resorbing activities of osteoclasts and prevented bone loss induced by estrogen deficiency but did not affect bone formation. OST-4077, an orally active selective human cathepsin K inhibitor, may have the therapeutic potential for the treatment of diseases characterized by excessive bone loss including osteoporosis.
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Amidas/farmacología , Amidas/uso terapéutico , Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Catepsinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Furanos/farmacología , Furanos/uso terapéutico , Osteoclastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores , Densidad Ósea/efectos de los fármacos , Resorción Ósea/metabolismo , Catepsina K , Catepsinas/genética , Clonación Molecular , Estrógenos/deficiencia , Femenino , Humanos , Ovariectomía , Hormona Paratiroidea/farmacología , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.
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Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Osteoclastos/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Butionina Sulfoximina/farmacología , Proteínas Portadoras/farmacología , Línea Celular , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Immunoblotting , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , FN-kappa B/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/citología , Oxidación-Reducción/efectos de los fármacos , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/metabolismoRESUMEN
Racemic fluids of chiral calamitic molecules are investigated with molecular dynamics simulations. In particular, the phase behavior as a function of density is examined for eight racemates. The relationship between chiral discrimination and orientational order in the phase is explored. We find that the transition from the isotropic phase to a liquid crystal phase is accompanied by an increase in chiral discrimination, as measured by differences in radial distributions. Among ordered phases, discrimination is largest for smectic phases with a significant preference for heterochiral contact within the layers.
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A spatial filter design method to reduce magnetic noise in the magnetocardiogram (MCG) is introduced. Based on the facts that external magnetic noise appearing on multichannel MCG sensors is independent of the cardiac signals and that there is strong spatial correlation among the channels, the independent component analysis (ICA) method was applied to extract the noise components from the measured MCG signals. After extraction of the noise components in a given time period using ICA, a spatial filter was made to reduce the noise components in subsequently acquired MCG signals. In experimental studies of nine healthy volunteers, the spatial filters improved the signal-to-noise ratio of the MCG signals by about 500% on average. This spatial filtering method can be used for measurements of MCG signals in a magnetically noisy environment.
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Pruebas de Función Cardíaca/métodos , Magnetismo , Procesamiento de Señales Asistido por Computador , Artefactos , HumanosRESUMEN
Refractory anemia with excess blasts in transformation (RAEB-T) is a subgroup of myelodysplastic syndrome (MDS) in which the bone marrow blast count ranges from 20% to 30%. The recently proposed World Health Organization Classification of Hematologic Malignancies eliminated this category from MDS by lowering the blast count cutoff for acute myeloid leukemia (AML) from 30% to 20%. However, MDS is distinguished from AML by a significant increase in apoptosis. To investigate the difference in apoptosis between RAEB-T, AML, and other categories of MDS, we prospectively analyzed fresh bone marrow samples using the Annexin V and mitochondrial potential assays. There was a significantly higher level of apoptosis in RAEB-T than in AML according to both assays, while no significant differences between RAEB-T and other categories of MDS were noted. The data suggest that RAEB-T is more likely to be an advanced stage of MDS and biologically different from AML.
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Anemia Refractaria con Exceso de Blastos/patología , Apoptosis , Leucemia Mieloide/patología , Enfermedad Aguda , Anemia Refractaria con Exceso de Blastos/metabolismo , Anexina A5/metabolismo , Antígenos CD34/metabolismo , Médula Ósea/patología , Estudios de Casos y Controles , Citometría de Flujo , Humanos , Leucemia Mieloide/metabolismo , Potenciales de la Membrana , Mitocondrias/metabolismo , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Estudios ProspectivosRESUMEN
The effects of food restriction on the serotonergic system were investigated immunohistochemically in both the midbrain and hypothalamic regions of rats. Rats were fed on a restricted feeding schedule consisting of half of the ad libitum quantity for 1, 2, 4 and 6 weeks and a free feeding schedule for 1 week. The optical density of serotonin-positive neurons in the raphe nuclei of the midbrain was found to be significantly lower in the 1 week-food restricted group than in the ad libitum fed control. In the hypothalamus, serotonin-positive neurons were observed in the 1 and 2 week food restricted groups but not in the 4 and 6 week-food restricted groups. This finding was confirmed by immunohistochemistry with tryptophan hydroxylase, a serotonin synthesizing enzyme. In this study, we provide morphological evidence that food restriction has a significant effect on the serotonergic system of the midbrain and hypothalamic regions and suggest some possibilities for the ectopic expression of serotonin-positive neurons after food restriction.
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Coristoma , Privación de Alimentos/fisiología , Hipotálamo/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Núcleos del Rafe/metabolismo , Serotonina/metabolismo , Animales , Peso Corporal/fisiología , Ingestión de Alimentos/fisiología , Hipotálamo/citología , Inmunohistoquímica , Masculino , Mesencéfalo/citología , Neuronas/citología , Núcleos del Rafe/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.
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Canales de Calcio/metabolismo , Cromograninas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Células COS , Canales de Calcio/inmunología , Bovinos , Cromogranina A , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/inmunología , Fracciones Subcelulares/metabolismoRESUMEN
The aim of this study was to investigate the electroacupuncture-related changes of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) in the brainstem of spontaneously hypertensive rats (SHR). We evaluated the changes of NADPH-d-positive neurons using a histochemical method and the changes of nNOS-positive neurons using an immunohistochemical method. The staining intensities of NADPH-d-positive neurons and nNOS-positive neurons were assessed in a quantitative fashion using a microdensitometrical method based on optical density by means of an image analyzer. The optical density of NADPH-d-positive neurons and nNOS-positive neurons of the Shinsu (BL23) and Choksamni (ST36) electroacupuncture groups were significantly decreased in most brainstem areas as compared to the normal and arbitrary groups, with the exception of the optical density of NADPH-d positive neurons in the prepositus nucleus as compared to the arbitrary group. The present results demonstrated that electroacupuncture changes the activity in the NO system in the brainstem of SHR and the site where electroacupuncture is administered is of importance for this effect.