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Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of Saccharomyces cerevisiae PSAT (ScPSAT) at a resolution of 2.8 Å. The results demonstrated that the ScPSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of ScPSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of ScPSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time.
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Saccharomyces cerevisiae , Transaminasas , Humanos , Estructura Molecular , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transaminasas/metabolismo , Fosfato de Piridoxal/metabolismo , Cristalografía por Rayos XRESUMEN
In this paper, a novel chip antenna and its function in wireless connectivity are presented for Bluetooth (BLT) earphones. The chip antenna is a metamaterial so compact (<λ/8), as the size of 4.9 × 13.0 × 2.0 mm3, that when it is mounted on the realistic PCB, it can be held in the enclosure of the BLT earphone. This setting does not degrade the resonance (S11 < −10 dB) of the proposed antenna. As two earphones in a pair are demanded to communicate with each other, one shares an RF signal with the other and they take turns as the master and slave. The received signal sensing is conducted with the latest model of human head-ear-phantom located between the earphones to mimic the real use-case and cross-head interference. Electromagnetic simulation of the antenna is done and verified by fabrication and measurement. Particularly, received-signal strength indications between the proposed antennas in the earphones are experimentally obtained as −67.5 dBm and −70 dBm without and with the head-ear-phantom, respectively, much greater than −120 dBm, the limit of detection, and implying acceptable connectivity and invulnerability over cross-head-interference problems.
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Fantasmas de Imagen , Simulación por Computador , HumanosRESUMEN
In this paper, a novel thin and flexible antenna is proposed for earbuds to gain an improvement in their wireless signal-sensing capability as a film-based artificial magnetic conductor (AMC) structure. As antenna designs for earbuds face challenges of being embedded beneath the top cover of the earbud, conformal to curved surfaces, and very close to metallic ground and touch-panel parts, as well as scarce degrees of freedom from feeding conditions and functional degradation by human tissue, unlike conventional techniques such as quasi quarter-wavelength radiators on LDS and epoxy molding compounds (relatively thick and pricy), an antenna of a metal pattern on a film is made with another film layer as the AMC to mitigate problems of the antenna in a small and curved space of an insert-molded wireless device. The antenna was designed, fabricated, and embedded in earbud mockups to work for the 2.4 GHz Bluetooth RF link, and its functions were verified by RF and antenna measurement, showing that it could overcome the limitations in impedance matching with only lumped elements and poor radiation by the ordinary schemes. The input reflection coefficient and antenna efficiency were 10 dB and 9% better than other methods. In particular, the on-film AMC antenna (OFAA) presents robustness against deterioration by the human tissue, when it is placed in the ear phantom at the workbench and implemented in an in situ test using a large zorb ball mimicking a realistic sensing environment. This yielded an RSSI enhancement of 20-30 dB.
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Tecnología Inalámbrica , Impedancia Eléctrica , Diseño de Equipo , Humanos , Fantasmas de ImagenRESUMEN
In this paper, an intuitive approach to assessing advantages of beamforming in 5G wireless communication is proposed as a novel try and practical demonstration of importance of alignment between the transmitter's and receiver's beams working in millimeter-wave frequency bands. Since the diffraction loss of millimeter-wave signals matters seriously in propagation, the effects of the misalignment and alignment between beams need to be checked for, which was conducted with a horn antenna and the 4 × 4 Butler matrix which mimic the relationship of the base station and handset antennas. Designing and using the microstrip-line and the substrate integrated waveguide (SIW) Butler matrices, RF-to-RF wireless connectivity between the horn and the microstrip line beamformer as case 1 and the horn and the SIW beamformer as case 2, concerning the changing angle of the beam from either of the two Butler matrices, was tested, showing over 12 dB enhancement in received power. This direct electromagnetic link test was accompanied by examining 64-QAM constellations for beam-angle changing from -30° to +30° for the two cases, where the error vector magnitude in the QAM-diagram becomes less than 10% by beam-alignment for the changing angle.
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OBJECTIVE: To test the hypothesis that Notch signalling plays a role in the pathogenesis of rheumatoid arthritis (RA) and to determine whether pharmacological inhibition of Notch signalling with γ-secretase inhibitors can ameliorate the RA disease process in an animal model. METHODS: Collagen-induced arthritis was induced in C57BL/6 or Notch antisense transgenic mice by immunisation with chicken type II collagen (CII). C57BL/6 mice were administered with different doses of inhibitors of γ-secretase, an enzyme required for Notch activation, at disease onset or after onset of symptoms. Severity of arthritis was monitored by clinical and histological scores, and in vivo non-invasive near-infrared fluorescence (NIRF) images. Micro-CT was used to confirm joint destruction. The levels of CII antibodies and cytokines in serum were determined by ELISA and bead-based cytokine assay. The expression levels of cytokines were studied by quantitative PCR in rheumatoid synovial fibroblasts. RESULTS: The data show that Notch signalling stimulates synoviocytes and accelerates their production of proinflammatory cytokines and immune responses involving the upregulation of IgG1 and IgG2a. Pharmacological inhibition of γ-secretase and antisense-mediated knockdown of Notch attenuates the severity of inflammatory arthritis, including arthritis indices, paw thickness, tissue damage and neutrophil infiltration, and reduces the levels of active NF-κB, ICAM-1, proinflammatory cytokines and matrix metalloproteinase-3 activity in the mouse model of RA. CONCLUSIONS: These results suggest that Notch is involved in the pathogenesis of RA and that inhibition of Notch signalling is a novel approach for treating RA.
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Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Citocinas/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Membrana Sinovial/inmunología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Citocinas/efectos de los fármacos , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Notch/antagonistas & inhibidores , Receptores Notch/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacosRESUMEN
Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.
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Artritis Reumatoide/genética , Artritis Reumatoide/terapia , Quitosano , Silenciador del Gen , Nanopartículas , ARN Interferente Pequeño/genética , Compuestos de Sulfhidrilo , Factor de Necrosis Tumoral alfa/genética , Animales , Artritis Experimental , Artritis Reumatoide/patología , Línea Celular , Quitosano/química , Modelos Animales de Enfermedad , Expresión Génica , Macrófagos/metabolismo , Masculino , Ratones , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Compuestos de Sulfhidrilo/química , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/sangreRESUMEN
The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aß), a key molecule that causes major neurotoxicity during the early stage of Alzheimer's disease (AD). However, despite its important role in Aß production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aß production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aß production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aß generation.
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Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/biosíntesis , Animales , Fibroblastos/metabolismo , Biblioteca de Genes , Humanos , Glicoproteínas de Membrana/genética , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados/metabolismo , Proteínas de Neoplasias/genética , Receptores Notch/metabolismoRESUMEN
Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.
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Indicán , Fallo Renal Crónico , Receptores de Hidrocarburo de Aril , Animales , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/metabolismo , Humanos , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/efectos de los fármacos , Ácido Araquidónico/metabolismo , Masculino , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos C57BL , Araquidonato 5-Lipooxigenasa/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Inmunidad EntrenadaRESUMEN
This study examined the influence of the N-methyl-D-aspartate receptor (NMDAR) on the modulation of related spinal signaling after electroacupuncture (EA) treatment in normal rats. Bilateral 2 Hz EA stimulations (1-2-3.0 mA) were delivered at acupoints corresponding to Zusanli (ST36) and Sanyinjiao (SP6) in men for 30 min. Thermal sensitization was strongly inhibited by EA, but this analgesia was reduced by preintrathecal injection of the NMDAR antagonist, MK801. Phosphorylation of the NMDAR NR2B subunit, cAMP response element-binding protein (CREB), and especially phosphatidylinositol 3-kinase (PI3K) were significantly induced by EA. However, these marked phosphorylations were not observed in MK801-pretreated rats. EA analgesia was reduced by preintrathecal injection with the calcium chelators Quin2 and TMB8, similar to the results evident using MK801. Phosphorylation of PI3K and CREB induced by EA was also inhibited by TMB8. Calcium influx by NMDAR activation may play an important role in EA analgesia of normal rats through the modulation of the phosphorylation of spinal PI3K and CREB.
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When we evaluated changes of glial fibrillary acidic protein (GFAP) and two glutamate transporter (GTs) by immunohistochemistry, expression of GFAP showed a significant increase in complete Freund's adjuvant (CFA)-injected rats; however, this expression was strongly inhibited by electroacupuncture (EA) stimulation. Robust downregulation of glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) was observed in CFA-injected rats; however, EA stimulation resulted in recovery of this expression. Double-labeling staining showed co-localization of a large proportion of GLAST or GLT-1 with GFAP. Using Western blot, we confirmed protein expression of two GTs, but no differences in the mRNA content of these GTs were observed. Because EA treatment resulted in strong inhibition of CFA-induced proteasome activities, we examined the question of whether thermal sensitivities and GTs expression could be regulated by proteasome inhibitor MG132. CFA-injected rats co-treated with EA and MG132 showed a significantly longer thermal sensitivity, compared with CFA-injected rats with or without MG132. Both EA and MG132 blocked CFA-induced GLAST and GLT-1 downregulation within the spinal cord. These results provide evidence for involvement of GLAST and GLT-1 in response to activation of spinal astrocytes in an EA antinociceptive effect. Antinociceptive effect of EA may be induced via proteasome-mediated regulation of spinal GTs.
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Objectives: Kidney transplant (KT) is the most effective treatment for end-stage renal disease. The immunosuppressant anti-thymocyte globulin (ATG) has been applied for induction therapy to reduce the risk of acute transplant rejection for patients at high immunological risk. Despite its putative role in replicative stress during immune reconstitution, the effects of ATG on T-cell immunosenescent changes remain to be understood. Methods: Phenotypic and functional features of senescent T cells were examined by flow cytometry in 116 healthy controls (HC) and 95 KT patients for comparative analysis according to ATG treatment and CMV reactivation. The TCR repertoire was analysed in peripheral blood mononuclear cells (PBMCs) of KT patients. Results: T cells of KT patients treated with ATG (ATG+) show typical immunosenescent features, accumulation of CD28-, CD85j+ or CD57+ T cells, and imbalance of functional T-cell subsets, compared with untreated KT patients (ATG-). Plasma IL-15 and CMV-IgG levels were higher in KT patients than in HCs, and the IL-15 level positively correlated with the frequency of CD28- T cells in KT patients. ATG+ patients had a higher prevalence of CMV reactivation, which is associated with an increased frequency of CD28- T cells. As a result, ATG+ patients had expanded CMV-specific T cells and decreased TCR diversity. However, proliferation, cytokine-producing capacity and polyfunctionality of T cells were preserved in ATG+ patients. Conclusion: Our findings suggest that ATG treatment contributes to the accumulation of senescent T cells, which may have lifelong clinical implications in KT patients. Thus, these patients require long-term and comprehensive immune monitoring.
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Existing studies examining the control of mesenchymal stem cell (MSC) differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry.
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Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Resistencia a la Tracción , Animales , Diferenciación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/fisiología , Conejos , Propiedades de SuperficieRESUMEN
Scleroderma is an autoimmune rheumatic disorder accompanied by severe fibrosis in skin and other internal organs. During scleroderma progression, resident fibroblasts undergo activation and convert to α-smooth muscle actin (α-SMA) expressing myofibroblasts (MFBs) with increased capacity to synthesize collagens and fibrogenic components. Accordingly, MFBs are a major therapeutic target for fibrosis in scleroderma and treatment with blocking MFBs could produce anti-fibrotic effects. TLY012 is an engineered human TNF-related apoptosis-inducing ligand (TRAIL) which induces selective apoptosis in transformed cells expressing its cognate death receptors (DRs). Here we report that TLY012 selectively blocks activation of dermal fibroblasts and induces DR-mediated apoptosis in α-SMA+ MFBs through upregulated DR5 during its activation. In vivo, TLY012 reverses established skin fibrosis to near-normal skin architecture in mouse models of scleroderma. Thus, the TRAIL pathway plays a critical role in tissue remodeling and targeting upregulated DR5 in α-SMA+ MFBs is a viable therapy for fibrosis in scleroderma.
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Actinas/genética , Dermis/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Esclerodermia Sistémica/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Actinas/metabolismo , Adulto , Anciano , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Diferenciación Celular , Colágeno/genética , Colágeno/metabolismo , Dermis/metabolismo , Dermis/patología , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , Miofibroblastos/metabolismo , Miofibroblastos/patología , Ingeniería de Proteínas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patología , Transducción de SeñalRESUMEN
Progressive renal failure causes uremia-related immune dysfunction, which features a chronic inflammatory milieu. Given the central role of end-stage renal disease (ESRD)-related immune dysfunction in the pathogenesis of cardiovascular diseases (CVDs), much attention has been focused on how uremic toxins affect cellular immunity and the mechanisms underlying pathogenesis of atherosclerosis in ESRD patients. Here, we investigated the characteristics of monocytes and CD4+ T cells in ESRD patients and the immune responses induced by indoxyl sulfate (IS), a key uremic toxin, in order to explore the pathogenic effects of these cells on vascular endothelial cells. In ESRD patients, monocytes respond to IS through the aryl hydrocarbon receptor (AhR) and consequently produce increased levels of TNF-α. Upon stimulation with TNF-α, human vascular endothelial cells produce copious amounts of CX3CL1, a chemokine ligand of CX3CR1 that is highly expressed on CD4+CD28-T cells, the predominantly expanded cell type in ESRD patients. A migration assay showed that CD4+CD28- T cells were preferentially recruited by CX3CL1. Moreover, activated CD4+CD28- T cells exhibited cytotoxic capability allowing for the induction of apoptosis in HUVECs. Our findings suggest that in ESRD, IS-mediated immune dysfunction may cause vascular endothelial cell damage and thus, this toxin plays a pivotal role in the pathogenesis of CVD.
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Linfocitos T CD4-Positivos/inmunología , Endotelio Vascular/patología , Indicán/toxicidad , Fallo Renal Crónico/inmunología , Adulto , Anciano , Apoptosis , Linfocitos T CD4-Positivos/efectos de los fármacos , Receptor 1 de Quimiocinas CX3C/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CXCL1/metabolismo , Endotelio Vascular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Indicán/metabolismo , Fallo Renal Crónico/patología , Masculino , Persona de Mediana Edad , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND AND AIMS: The pathogenesis of coronary artery disease (CAD) is closely associated with chronic inflammatory processes. CD8(+) T cells are a key participant in the pathogenesis of atherosclerosis, the major cause of CAD; however, it remains unclear which CD8(+) T-cell subset is responsible. We investigated the immunological features of CD8(+) T cells expressing low and high levels of the IL-6 receptor α chain (IL-6Rα), a cytokine known to play a key role in cardiovascular diseases. METHODS: The expression of IL-6Rα on CD8(+) T cells and its association with plasma levels of soluble components of the IL-6/IL-6Rs as well as with clinical parameters were analyzed using FACS analysis and ELISA of CAD patients and age-matched healthy controls (HCs). Immunological characteristics of CD8(+) T cells expressing low and high levels of IL-6Rα (CD8(+)IL-6Rα(low or high)) were examined by in vitro culture and intracellular FACS analysis. RESULTS: CAD patients had higher frequencies of circulating CD8(+)IL-6Rα(low) effector memory (EM) T cells compared with HCs (median frequency; 74.59% vs. 60.09%, p = 0.0158). Expanded CD8(+)IL-6Rα(low) T cells positively correlated with the frequency of senescent, cytotoxic CD8(+)CD57(+) T cells (r = 0.6655, p < 0.0001) and plasma IL-6 level (r = 0.3995, p = 0.0432) in CAD patients. Loss of IL-6Rα expression on CD8(+) T cells was induced by the combination of IL-6 and IL-15 with accompanying TCR-independent proliferation (p = 0.0101). Moreover, these CD8(+)IL-6Rα(low) T cells had features of type 1 cytotoxic CD8(+) T cells. CONCLUSIONS: Our findings suggest the possible involvement of expanded CD8(+)IL-6Rα(low) EM T cells in CAD through their pro-inflammatory and highly cytotoxic capacities.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Enfermedad de la Arteria Coronaria/genética , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Anciano , Separación Celular , Femenino , Citometría de Flujo , Humanos , Inflamación , Interleucina-15/metabolismo , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Presenilin/gamma-secretase is a membrane-associated protease that cleaves within the transmembrane region of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta) whose deposition in the brain is a characteristic of Alzheimer's disease (AD). Calsenilin, a calcium binding protein that has been shown to interact with the C-termini of both presenilin 1 (PS1) and presenilin 2 (PS2), appears to play a role in transcriptional regulation and apoptosis and to bind to A-type voltage-gated potassium channels. Here, we report that overexpression of calsenilin enhanced gamma-secretase activity in cells. The effect of calsenilin on the gamma-cleavage of substrates was blocked by the selective gamma-secretase inhibitor L-685,458. We also employed a cellular gamma-cleavage GFP-reporter assay to demonstrate the effect of calsenilin on gamma-secretase activity. To establish a direct role for calsenilin in regulating gamma-secretase activity, we incubated purified calsenilin with isolated membrane fractions and found increased Abeta production in a cell free system. These data suggest that calsenilin may be one of the regulatory factors for gamma-secretase.
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Proteínas de Unión al Calcio/biosíntesis , Endopeptidasas/metabolismo , Proteínas Represoras/biosíntesis , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/biosíntesis , Animales , Ácido Aspártico Endopeptidasas , Sistema Libre de Células , Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas de Interacción con los Canales Kv , TransfecciónRESUMEN
PURPOSE: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death. MATERIALS AND METHODS: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2x10(5) cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200-300 cGy/min. The cells were treated with 0.25 microM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins. RESULTS: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation. CONCLUSIONS: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.
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Apoptosis/efectos de la radiación , Caspasas/metabolismo , Inhibidores Enzimáticos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Membranas Mitocondriales/fisiología , Quinonas/farmacología , Benzoquinonas , Activación Enzimática , Fase G2 , Humanos , Lactamas Macrocíclicas , Potenciales de la Membrana , Mitosis/fisiología , Mitosis/efectos de la radiación , Rifabutina/análogos & derivados , Células Tumorales Cultivadas , Rayos XRESUMEN
Notch pathway plays a pivotal role in synoviocytes involved in progression of rheumatoid arthritis (RA). Herein, we designed the Notch1 targeting siRNA delivery nanoparticles (siRNA-NPs) in order to confirm the anti-inflammatory effect in collagen-induced arthritis (CIA) model. The siRNA-NPs were successfully produced by encapsulating polymerized siRNA (poly-siRNA) into thiolated glycol chitosan (tGC) nanoparticles in aqueous condition. The in vitro Notch1 inhibition of siRNA-NPs in murine macrophage cell (RAW 264.7) was confirmed using confocal microscopy and real time PCR. Fluorescently labeled siRNA-NPs were successfully transfected in RAW 264.7 and modulated the expression of Notch1 in mRNA level. For in vivo study, siRNA-NPs exhibited the higher targeting efficiency in the arthritic joins of CIA mice, confirmed by the near-infrared fluorescence (NIRF) imaging. Furthermore, inhibition of Notch1 with siRNA-NPs resulted in retarded progression of inflammation, bone erosion, and cartilage damage in CIA mice. Novel Notch1 targeting siRNA delivery system of siRNA-NPs showed effective RA treatment by suppressing Notch1 signaling pathway without undesirable severe toxicity. Thus, Notch1 inhibiting siRNA-NPs demonstrated the great potential in RA therapeutics that was hard to be achieved using conventional drugs.
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Artritis Reumatoide/terapia , Técnicas de Transferencia de Gen , ARN Interferente Pequeño/farmacología , Receptor Notch1/efectos de los fármacos , Animales , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/patología , Huesos/patología , Cartílago/patología , Quitosano , Colágeno , Progresión de la Enfermedad , Terapia Genética , Humanos , Ratones , Nanopartículas , Células RAW 264.7 , TransfecciónRESUMEN
Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.
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Adenoviridae/genética , Enfermedad de Charcot-Marie-Tooth/enzimología , Animales , Axones/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Vectores Genéticos , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/enzimología , Mutación Missense , Fibras Nerviosas Mielínicas/enzimología , Especificidad de Órganos , Nervios Periféricos/enzimología , Nervios Periféricos/patologíaRESUMEN
Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium-triggered neuronal apoptosis, and γ-secretase activity. Notch is a cell surface receptor that regulates cell-fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ-secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic-like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ-secretase cleavage of Notch and to contribute to cell death under ischemia-like conditions. The inhibition of γ-secretase activity and a presenilin deficiency were both found to protect against calsenilin-mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ-secretase cleavage of Notch.