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BACKGROUND: Self-efficacy is crucial in improving medical students' communication skills. This study aims to clarify where medical students' self-efficacy is greatest following an interview with a simulated patient and subsequent feedback. METHODS: A total of 162 medical students (109 men, 53 women) in their fourth or fifth year at a university in Japan participated in this study. The degree of self-efficacy in medical interviewing was measured before and after a medical interview with a simulated patient, and after the subsequent feedback session. RESULTS: ANOVA analysis revealed that self-efficacy for medical interviews was higher after both the interview and the feedback session than before the interview. Among all three time points, self-efficacy was highest after the feedback session. CONCLUSIONS: Feedback following a simulated interview with a simulated patient is important to improve the self-efficacy of medical students when learning medical interviewing skills.
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Educación de Pregrado en Medicina , Estudiantes de Medicina , Competencia Clínica , Comunicación , Retroalimentación , Femenino , Humanos , Japón , Masculino , AutoeficaciaRESUMEN
BACKGROUND: Professional identity formation is nurtured through socialization, driven by interaction with role models, and supported through early clinical exposure (ECE) programmes. Non-healthcare professionals form part of the hospital community but are external to the culture of medicine, with their potential as role models unexplored. We employed text mining of student reflective assignments to explore the impact of socialization with non-healthcare professionals during ECE. METHODS: Assignments from 259 first-year medical students at Fukushima Medical University, Japan, underwent hierarchical cluster analysis. Interrelationships between the most-frequently-occurring words were analysed to create coding rules, which were applied to elucidate underlying themes. RESULTS: A shift in terms describing professional characteristics was detected, from "knowledge/skill" towards "pride [in one's work]" and "responsibility". Seven themes emerged: contribution of non-healthcare professionals, diversity of occupation, pride, responsibility, teamwork, patient care and gratitude. Students mentioning 'contribution of non-healthcare professionals' spoke of altruistic dedication and strong sense of purpose. These students expressed gratitude towards non-healthcare professionals for supporting clinical work, from a doctor's perspective. CONCLUSION: Socialization with non-healthcare professionals provides important insights into the hospital working environment and cultural working norms. Through role modelling altruism and responsibility, non-healthcare professionals positively influenced student professional identity formation, promoting self-conceptualisation as a doctor.
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Estudiantes de Medicina , Minería de Datos , Humanos , Japón , Personal de Hospital , Identificación SocialRESUMEN
AIMS: The aim of this study was to investigate the effects of a single green tea (GT), administered concomitantly or 1 hour before nadolol intake on nadolol pharmacokinetics. METHODS: In a randomized 3-phase crossover study, 11 healthy volunteers received an oral administration of nadolol with, or 1 hour after preingestion of brewed GT, or with water in a volume of 150 mL. RESULTS: Geometric mean ratio with 90% confidence interval for nadolol AUC0-48 was 0.371 (0.303-0.439) with concomitant GT. In addition, ingestion of GT 1 hour before nadolol administration resulted in a significant reduction of nadolol AUC0-48 with geometric mean ratio of 0.536 (0.406-0.665). There were no differences in time to maximal plasma concentration and renal clearance of nadolol among groups. CONCLUSION: These results suggest that single concomitant ingestion of GT substantially decreases plasma concentrations of nadolol. Moreover, the reduction in nadolol bioavailability could persist for at least 1 hour after drinking a cup of GT.
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Catequina , Nadolol , Catequina/análisis , Estudios Cruzados , Ingestión de Alimentos , Voluntarios Sanos , Humanos , TéRESUMEN
Glucocorticoid (GC) resistance is associated with poor response to the following chemotherapy in lymphoid malignancies, such as lymphoma and leukemia. However, it remains unclear whether GCs interfere with the cytotoxic effects of anti-cancer drugs on GC-resistant cells. In this study, we examined whether GCs affected the sensitivities to vincristine (VCR)/doxorubicin (DOX) and the expression of drug transporters in GC-resistant cells. The dexamethasone (DEX)/prednisolone (PSL)-resistant lymphoid and non-lymphoid cell lines Raji and HL60 were cultured with DEX for 7 days and then treated with VCR or DOX for 3 days. Seven days of DEX treatment increased the IC50s of both VCR and DOX in Raji cells but not in HL60 cells. The mRNA and protein expression levels of organic cation/carnitine transporter (OCTN) 2, one of the drug uptake transporters expressed in both cell lines, were decreased only in Raji cells. When Raji cells were cultured with PSL, the IC50 of DOX but not VCR increased as the expression of OCTN2 decreased. No significant increases in efflux transporter expression were induced by DEX or PSL. When siRNA against OCTN2 was introduced into Raji cells, the IC50 of DOX but not VCR increased significantly. These data suggested that both DEX and PSL decreased the sensitivity of the DEX/PSL-resistant Raji cells to DOX, a change that was at least partially due to reductions in OCTN2. Thus, the continuous usage of GCs may interfere with the effects of chemotherapy on GC-resistant lymphoid cells.
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Dexametasona/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Glucocorticoides/farmacología , Linfocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Prednisolona/farmacología , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Células HL-60 , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Leucemia/patología , Linfocitos/patología , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/patologíaRESUMEN
In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8-induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.
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Quimiotaxis de Leucocito , MicroARNs/inmunología , Síndromes Mielodisplásicos/inmunología , Activación Neutrófila , Neutrófilos/fisiología , Anciano , Anciano de 80 o más Años , Proliferación Celular , Quimiotaxis/inmunología , Femenino , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HL-60 , Humanos , Interleucina-8/inmunología , Masculino , MicroARNs/genética , MicroARNs/fisiología , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Neutrófilos/inmunología , ARN Interferente Pequeño , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/inmunología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/inmunologíaRESUMEN
PURPOSE: The objective of this study is to assess the effects of green tea and its major catechin component, (-)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. METHODS: The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. RESULTS: EGCG inhibited diclofenac 4'-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 µM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration-time curve (AUC0-∞) of fluvastatin were 0.993 (0.963-1.024, vs. brewed green tea) and 0.977 (0.935-1.020, vs. GTE). CONCLUSIONS: Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.
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Catequina/análogos & derivados , Citocromo P-450 CYP2C9/metabolismo , Ácidos Grasos Monoinsaturados/farmacocinética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Indoles/farmacocinética , Té , Adulto , Antiinflamatorios no Esteroideos/farmacocinética , Catequina/análisis , Catequina/sangre , Catequina/farmacocinética , Catequina/farmacología , Estudios Cruzados , Diclofenaco/farmacocinética , Ácidos Grasos Monoinsaturados/sangre , Femenino , Fluvastatina , Interacciones Alimento-Droga , Voluntarios Sanos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/sangre , Indoles/sangre , Masculino , Té/química , Adulto JovenRESUMEN
PURPOSE: The aim of the present study is to investigate a possible role of a single dose of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, for the pharmacokinetic interaction between green tea and nadolol in humans. METHODS: In a randomized three-phase crossover study, 13 healthy volunteers received single doses of 30 mg nadolol orally with water (control), or an aqueous solution of EGCG-concentrated green tea extract (GTE) at low or high dose. Plasma concentrations and urinary excretion of nadolol were determined up to 48 h. In addition, blood pressure and pulse rate were monitored. In vitro transport kinetic experiments were performed using human embryonic kidney 293 cells stably expressing organic anion transporting polypeptide (OATP)1A2 to evaluate the inhibitory effect of EGCG on OATP1A2-mediated substrate transport. RESULTS: Single coadministration of low and high dose GTE significantly reduced the plasma concentrations of nadolol. The geometric mean ratios with 90% CI for area under the plasma concentration-time curves from 0 to infinity of nadolol were 0.72 (0.56-0.87) for the low and 0.60 (0.51-0.69) for the high dose. There were no significant differences in Tmax, elimination half-life, and renal clearance between GTE and water phases. No significant changes were observed for blood pressure and pulse rate between phases. EGCG competitively inhibited OATP1A2-mediated uptake of sulphobromophthalein and nadolol with Ki values of 21.6 and 19.4 µM, respectively. CONCLUSIONS: EGCG is suggested to be a key contributor to the interaction of green tea with nadolol. Moreover, even a single coadministration of green tea may significantly affect nadolol pharmacokinetics.
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Antagonistas Adrenérgicos beta/farmacocinética , Antioxidantes/farmacología , Camellia sinensis , Catequina/análogos & derivados , Nadolol/farmacocinética , Extractos Vegetales/farmacología , Antagonistas Adrenérgicos beta/sangre , Antagonistas Adrenérgicos beta/orina , Adulto , Antioxidantes/análisis , Proteínas Sanguíneas/metabolismo , Catequina/análisis , Catequina/farmacología , Estudios Cruzados , Interacciones Farmacológicas , Femenino , Células HEK293 , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Nadolol/sangre , Nadolol/orina , Transportadores de Anión Orgánico , Extractos Vegetales/análisis , Unión Proteica , Adulto JovenRESUMEN
BACKGROUND: Pediatric apheresis for peripheral blood stem cell transplantation should be carried out with due concern for low corporeal blood volume and vulnerability to hypocalcemia-related complications, hypovolemic shock, and hypervolemic cardiac overload. STUDY DESIGN AND METHODS: We retrospectively investigated a total of 267 apheresis procedures from 1990 to 2013 on 93 children between 0 and 10 years old, including 89 patients and 4 healthy donors, with body weights of 6.3 to 44.0 kg. RESULTS: The median CD34+ cell yield per apheresis procedure was 2.3 × 106 CD34+ cells/kg (0.2-77.9 × 106 CD34+ cells/kg). Adverse events occurred in 11.6% of procedures (n = 31), including mild perivascular pain (n = 12), emesis (n = 9), hypotension (n = 3), urticaria (n = 2), numbness (n = 2), chest pain (n = 1), facial flush (n = 1), and abdominal pain (n = 1). Among hypotensive events, shock in a 9.6 kg one-year-old boy required emergency treatment in 1996. Thereafter, we adopted continuous injection of calcium gluconate, ionized calcium monitoring, central venous catheter access and circuit priming with albumin in addition to concentrated red cells. Since then we have had fewer complications: 16.4% per apheresis during 1990-1997 versus 5.8% during 1998-2013. No healthy pediatric donors suffered from any late-onset complications related to apheresis or G-CSF administration. CONCLUSION: By employing appropriate measures, peripheral blood stem cell apheresis for small children can have an improved safety profile, even for children weighing <10 kg.
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Eliminación de Componentes Sanguíneos/métodos , Movilización de Célula Madre Hematopoyética/métodos , Atención al Paciente/métodos , Células Madre de Sangre Periférica/citología , Antígenos CD34/metabolismo , Eliminación de Componentes Sanguíneos/efectos adversos , Donantes de Sangre , Presión Sanguínea , Peso Corporal/efectos de los fármacos , Calcio/administración & dosificación , Calcio/farmacología , Niño , Preescolar , Suplementos Dietéticos , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Dolor/etiología , Células Madre de Sangre Periférica/efectos de los fármacosRESUMEN
Accurate and efficient self-assessment is a critical skill for medical students to develop as part of their professional development. Along with clinical training reform at Fukushima Medical University, rubric-based student self-assessment and teacher assessment of students' performance using our proposed assessment tool, which includes several aspects of clinical skills and abilities, was initiated to improve the clinical clerkship process. To investigate how students identified their weaknesses and strengths, we analyzed the results of 119 fourth-year medical students' self-assessment and corresponding teacher assessment. Our study revealed substantial consistency between student self-assessment and teacher assessment, despite some overestimation and underestimation in student self-assessments. Students who incorrectly assess themselves require a variety of feedback to increase their self-efficacy and self-confidence, as well as to identify their weaknesses.
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Prácticas Clínicas , Estudiantes de Medicina , Humanos , Autoevaluación (Psicología) , Competencia Clínica , Prácticas Clínicas/métodosRESUMEN
Lisinopril, a highly hydrophilic long-acting angiotensin-converting enzyme inhibitor, is frequently prescribed for the treatment of hypertension and congestive heart failure. Green tea consumption may reduce the risk of cardiovascular outcomes and total mortality, whereas green tea or its catechin components has been reported to decrease plasma concentrations of a hydrophilic ß blocker, nadolol, in humans. The aim of this study was to evaluate possible effects of green tea extract (GTE) on the lisinopril pharmacokinetics. In an open-label, randomized, single-center, 2-phase crossover study, 10 healthy subjects ingested 200 mL of an aqueous solution of GTE containing ~ 300 mg of (-)-epigallocatechin gallate, a major catechin component in green tea, or water (control) when receiving 10 mg of lisinopril after overnight fasting. The geometric mean ratio (GTE/control) for maximum plasma concentration and the area under the plasma concentration-time curve of lisinopril were 0.289 (90% confidence interval (CI) 0.226-0.352) and 0.337 (90% CI 0.269-0.405), respectively. In contrast, there were no significant differences in time to reach maximum lisinopril concentration (6 hours in both phases) and renal clearance of lisinopril (57.7 mL/minute in control vs. 56.9 mL/minute in GTE). These results suggest that the extent of intestinal absorption of lisinopril was significantly impaired in the presence of GTE, whereas it had no major effect on the absorption rate and renal excretion of lisinopril. Concomitant use of lisinopril and green tea may decrease oral exposure to lisinopril, and therefore result in reduced therapeutic efficacy.
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Catequina/análogos & derivados , Interacciones Alimento-Droga , Lisinopril/farmacocinética , Té/química , Administración Oral , Adulto , Catequina/administración & dosificación , Catequina/farmacocinética , Estudios Cruzados , Ayuno , Femenino , Voluntarios Sanos , Humanos , Absorción Intestinal , Lisinopril/administración & dosificación , Masculino , Adulto JovenRESUMEN
BACKGROUND: pIREShyg2 has been widely used as a bicistronic expression vector. However, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA with a premature termination codon (PTC). In mammalian cells, the induction of NMD requires either a long 3'UTR or the presence of an exon-junction complex downstream of a PTC. The efficiency of NMD is greater when a PTC generates longer 3'UTR. pIREShyg2 provides the first cistron gene with a long 3'UTR consisting of a downstream intervening sequence (IVS), an internal ribosomal entry site (IRES) and the second cistron. Therefore, we hypothesized that the first cistron genes in pIREShyg2 are sensitized to NMD, which affects their expression levels. To examine this hypothesis, cDNAs encoding human granulocyte-macrophage colony-stimulating factor receptor beta chain (betac) and its splice variant (betac79), in which the retention of a 79-base intron caused a frameshift generating 18 PTCs, were cloned into pIREShyg2 and stably expressed in a murine cell line, Ba/F3. RESULTS: Compared with wild-type betac, the mRNA levels of betac79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of betac79 than wild-type betac. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type betac and betac79, suggesting that the NMD is attributable to a structural determinant in pIREShyg2. The elimination of the intron and the proximal 3' 17 PTCs did not alter the greater effects of translation inhibition on betac79, suggesting that the first PTC, which determines 3'UTR length, was sufficient to enhance NMD efficiency. Thus, transcripts of PTC-harboring genes with longer 3'UTR are more efficiently degraded by the vector-dependent NMD than those of wild-type genes with relatively shorter 3'UTR, resulting in minimized expression of truncated mutants. CONCLUSIONS: We conclude that pIREShyg2, which sensitizes its bicistronic transcripts to NMD, may be useful for studying NMD but should be avoided when maximum expressions of PTC-harboring genes are required.
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Codón sin Sentido , Vectores Genéticos , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Empalme Alternativo , Línea Celular , Mutación del Sistema de Lectura , Técnicas de Silenciamiento del Gen , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Intrones , ARN Helicasas , Transactivadores/deficiencia , Transactivadores/genética , Transactivadores/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVES: To clarify (1) the prevalence and associating factors of work-life conflict (WLC);(2) the details of gender-based discrimination;and (3) the association between WLC and gender-based discrimination among various professionals in a medical university organization. METHODS: This cross-sectional study, conducted in 2017, included all employees working at a public medical university and two affiliated hospitals that lie in provincial cities in Japan. The outcome of interest was time-based WLC in the work-to-family or family-to-work direction, measured with a shortened version of an existing scale. Gender-based discrimination was measured according to a three-point scale. RESULTS: Among the 3,347 employees, complete data sets were available for 2,285 (complete response rate, 68.3%). Of these, approximately 30% of respondents had perceived WLC. Multivariable logistic regression analysis showed that faculty members, nurses, and employees between 30 and 39 years old had a greater risk of WLC regardless of gender. Men were more likely to perceive gender-based discrimination in the contents of their work and the number of incidental tasks, while women were more likely to perceive discrimination with promotions and evaluation of academic achievements. Both men and women respondents who perceived gender-based discrimination had an increased risk of WLC. CONCLUSIONS: When promoting organizational well-being in a medical university, increased attention should be paid to faculty members, nurses and employees between 30 and 39 years old, as they have a greater risk of WLC. Our results also suggest that promoting gender equality is important to help achieve appropriate work-life balance.
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Sexismo , Equilibrio entre Vida Personal y Laboral , Adulto , Anciano , Estudios Transversales , Femenino , Equidad de Género , Hospitales Universitarios , Humanos , Modelos Logísticos , Masculino , Persona de Mediana EdadRESUMEN
In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca(2+) signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na(+)/Ca(2+) exchangers (NCX). The fluorometric study showed that the intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased by reducing extracellular Na(+) concentration ([Na(+)](o)) in tendon fibroblasts. Selective NCX inhibitors, KB-R7943 and SEA0400, both attenuated [Na(+)](o)-dependent [Ca(2+)](i) elevation and the resting [Ca(2+)](i) in tendon fibroblasts. RT-PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound-healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB-R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast-mediated collagen-matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca(2+) influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.
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Tendón Calcáneo/fisiología , Movimiento Celular/fisiología , Fibroblastos/fisiología , Contracción Muscular/fisiología , Intercambiador de Sodio-Calcio/fisiología , Tendón Calcáneo/citología , Tendón Calcáneo/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Wistar , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiourea/análogos & derivados , Tiourea/farmacología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
Survivin, which is a member of the inhibitor of apoptosis protein family, was found originally in immature cells and cancer cells but not in non-neoplastic adult tissues. The subsequent identification of four other alternative splice variants that possess distinct functions and localizations suggested the diverse roles of survivin isoforms. An unspecified isoform of survivin was found recently to be induced in terminally differentiated neutrophils by cytokines that prolong the neutrophil lifespan, such as GM-CSF and G-CSF, suggesting the importance of survivin in blocking apoptosis in neutrophils. To examine the mechanism by which survivin inhibits neutrophil apoptosis, we attempted to induce survivin by GM-CSF/G-CSF in an HL60 cell line that was differentiated into neutrophils by all-trans retinoic acid and DMSO and freshly isolated human neutrophils. The antiapoptotic isoform "Survivin," which was decreased during differentiation, was re-induced by GM-CSF in neutrophil-like, differentiated HL60. In contrast, in normal neutrophils, survivin mRNA was observed to increase spontaneously after 24 h incubation, and no additional elevation was induced by GM-CSF/G-CSF, which exerted their antiapoptotic effects on the neutrophils in 6 h, despite the lack of survivin induction. PCR and Western blotting detected Survivin-2 alpha, a dominant-negative of antiapoptotic Survivin, with no other isoforms in the freshly isolated or incubated neutrophils. Our study revealed that the expressed isoforms and the response to GM-CSF were different between the HL60-derived and normal neutrophils, which predominantly expressed Survivin-2 alpha, not likely involved in apoptosis inhibition by GM-CSF/G-CSF.
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Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neutrófilos/efectos de los fármacos , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Secuencia de Bases , Diferenciación Celular , Dimetilsulfóxido/farmacología , Exones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Genes Dominantes , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células HL-60/citología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Leucemia Promielocítica Aguda/patología , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neutrófilos/citología , Neutrófilos/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Survivin , Tretinoina/farmacologíaRESUMEN
High-mobility group AT-hook 2 (HMGA2) is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by MIRlet-7 and the polycomb-recessive complex 2 (PRC2) including EZH2. The HMGA2 messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of Ezh2 promotes the progression of severe myelofibrosis in JAK2V617F mice with upregulation of several oncogenes such as Hmga2. However, the direct role of HMGA2 in the pathogenesis of MPNs remains unknown. To clarify the impact of HMGA2 on MPNs carrying the driver mutation, we generated ΔHmga2/JAK2V617F mice overexpressing Hmga2 due to deletion of the 3' untranslated region. Compared with JAK2V617F mice, ΔHmga2/JAK2V617F mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. ΔHmga2/JAK2V617F cells showed a greater repopulating ability that reproduced the severe MPN compared with JAK2V617F cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of JAK2V617F erythroblasts that may worsen anemia. Relative to JAK2V617F hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in ΔHmga2/JAK2V617F HSPCs overlapped with those derepressed by Ezh2 loss in JAK2V617F/Ezh2Δ/Δ HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of Hmga2 ameliorated anemia and splenomegaly in JAK2V617F/Ezh2Δ/wild-type mice, and MIRlet-7 suppression and PRC2 mutations correlated with the elevated HMGA2 mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.
Asunto(s)
Granulocitos/efectos de los fármacos , Síndromes Mielodisplásicos/sangre , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Puromicina/farmacología , Estudios de Casos y Controles , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genéticaRESUMEN
We recently identified a reduction in the neutrophil surface expression of common beta chain (beta c) of the receptor for granulocyte macrophage-colony stimulating factor (GM-CSF) in the patients with myelodysplastic syndromes (MDS). To determine the etiology of the impaired beta c expression, beta c mRNA from neutrophilic granulocytes of MDS patients and healthy controls was analyzed by a combination of direct reverse transcriptase-polymerase chain reaction-based single-strand conformational polymorphism and sequencing. Nine different beta c transcripts were detected, but none was specific for MDS. However, one of the transcripts (beta c79) containing a 79-base intron insertion between exons V and VI was significantly increased in MDS. This 27-kd isoform consisted of the beta c N-terminal 182 amino acids followed by a new 84-amino-acid sequence. beta c79 was overexpressed in all MDS subtypes. No genomic mutations were detected within the intron or at the intron/exon boundaries. The isoform is predominantly located in the cytoplasm by Western blot analysis and was unable to generate high-affinity binding sites or transduce a signal for proliferation when coexpressed with the receptor for human GM-CSF alpha chain. Our study suggests that the accumulation of the abnormal beta c transcripts with intron V retention results in the reduction in cell-surface expression of beta c observed in MDS.
Asunto(s)
Intrones , Síndromes Mielodisplásicos/genética , Neutrófilos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Adulto , Anciano , Proliferación Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patología , Neutrófilos/patología , Polimorfismo Genético , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismoRESUMEN
Although increased TNF-α has been considered to cause ineffective hematopoiesis in myelodysplastic syndromes (MDS), the mechanisms of TNF-α elevation are not known. We recently found that c-Fos mRNA stabilization under translation-inhibiting stimuli was impaired in MDS-derived neutrophilic granulocytes. In the current study, we identified overexpression of c-Fos-targeting miR-34a and miR-155 as the cause of impairment. Expression levels of miR-34a but not miR-155 inversely correlated with ratios of c-Fos-positive cells in MDS-derived CD16+ neutrophils (r = -0.618, P<0.05), which were analyzed by flow cytometry. Among the seventeen patients, c-Fos was detectable in less than 60% of CD16+ cells in eight patients (Group A), while five (Group B) expressed c-Fos in more than 80% of CD16+ cells, which was consistent with the controls (88.6 ± 7.8%). Group A-derived granulocytes secreted more TNF-α in response to 1 µM LPS for 3 hours (735.4 ± 237.5 pg/mL) than Group B (143.5 ± 65.7 pg/mL, P<0.05) and healthy controls (150.8 ± 91.5 pg/mL, P<0.05). Knockdown of c-Fos in neutrophil-like differentiated HL60 increased the binding of NF-κB p65 to the promoter region of TNF-α DNA. Thus, c-Fos reduction via overexpression of miR-34a contributes to TNF-α overproduction under inflammatory stimuli in MDS.