RESUMO
BACKGROUND: It is useful to advance simulated patient (SP) participation in teaching to improve the communication skills of medical students, so this study aims to explore the current state of Chinese mainland SP education. METHODS: A cross sectional survey was designed utilizing well defined quantitative research methods and descriptive statistics. The questionnaire sought information which elucidated the current status of SP-based education, the origin of SP-based learning, SP training, challenges of this learning strategy and future developments. Questionnaires were distributed to 79 medical colleges in mainland China, and 68 were returned. Of these, 64 constituted valid responses (81%). RESULTS: The number of SP-based education activities in medical colleges offering 5-yearã7-year and 8-year clinical medicine programs was significantly higher than that in medical colleges which offered only a single 5-year program (p < 0.01). Communication skills training accounted for 73% of the content of SP-based learning activities, and was expected to rise in the future to 90%, in response to a need to improve doctor-patient relationships. Persons recruited as 'simulated patients' included students (21% of the total), residents (49%), medical staff (15%) and teaching staff (14%). Colleges, planning a SP-based education program, preferred teachers (80%) and students (55%) to assume 'simulated patient' roles. In objective structured clinical education (OSCE) scenarios, co-scoring by both SPs and teachers featured more highly in the 'consultation' station and 'doctor-patient communication' station. A number of factors were identified as hindering future development and implementation of SP-based learning including budget restraints, SP selection and training. CONCLUSIONS: SP-based learning programs offer clear benefits for improving the clinical education of medical students and their communication skills. The main obstacles to achieving more widespread and higher quality SP-based education are insufficient funding and the lack of standardized training and performance evaluation processes for simulated patients. Medical colleges should consider reducing the proportion of students and teachers acting as SPs, and attract more citizens to participate in SP-based learning activities. Formalised training and evaluation of SPs performance are necessary to establish a 'standard simulated patient' for a particular medical discipline, thus improving SP-based activities and student learning.
Assuntos
Estudantes de Medicina , Competência Clínica , Comunicação , Estudos Transversais , Humanos , Simulação de Paciente , Inquéritos e Questionários , UniversidadesRESUMO
The COVID-19 pandemic forced many educational institutions to turn to electronic learning to allow education to continue under the stay-at-home orders/requests that were commonly instituted in early 2020. In this cross-sectional study, we evaluated the effects of the COVID-19 pandemic on medical education in terms of students' attitudes toward online classes and their online accessibility; additionally, we examined the impacts of any disruption caused by the pandemic on achievement test performance based on the test results. The participants were 674 students (412 in pre-clinical, 262 in clinical) at Juntendo University Faculty of Medicine; descriptive analysis was used to examine the respondents' characteristics and responses. The majority of respondents (54.2%) preferred asynchronous classes. Mann-Whitney U tests revealed that while pre-clinical students preferred asynchronous classes significantly more than clinical students (39.6%, p < .001), students who preferred face-to-face classes had significantly higher total achievement test scores (U = 1082, p = .021, r = .22). To examine the impacts of pandemic-induced changes in learning, we conducted Kruskal-Wallis tests and found that the 2020 and 2021 scores were significantly higher than those over the last three years. These results suggest that while medical students may have experienced challenges adapting to electronic learning, the impact of this means of study on their performance on achievement tests was relatively low. Our study found that if possible, face-to-face classes are preferable in an electronic learning environment. However, the benefit of asynchronous classes, such as those that allow multiple viewings, should continue to be recognized even after the pandemic.
Assuntos
COVID-19/epidemiologia , Educação a Distância/métodos , Educação Médica/métodos , Estudantes de Medicina/psicologia , Adulto , COVID-19/psicologia , Instrução por Computador , Estudos Transversais , Escolaridade , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Japão , Masculino , Adulto JovemRESUMO
Chronic magnesium (Mg) deficiency induces and exacerbates various cardiovascular diseases. We previously investigated the mechanisms underlying decline in cardiac function caused by chronic Mg deficiency and the effectiveness of Mg supplementation on this decline using the Langendorff-perfused isolated mouse heart model. Herein, we used the Langendorff-perfused isolated rat heart model to demonstrate the chronic Mg-deficient rats (Mg-deficient group) had lower the heart rate (HR) and left ventricular pressure (LVDP) than rats with normal Mg levels (normal group). Furthermore, decline in cardiac function due to hypoxia/reoxygenation injury was significantly greater in the Mg-deficient group than in the normal group. Experiments on mitochondrial permeability transition pore (mPTP) using isolated mitochondria revealed that mitochondrial membrane was fragile in the Mg-deficient group, implying that cardiac function decline through hypoxia/reoxygenation injury is associated with mitochondrial function. Mg supplementation for chronic Mg-deficient rats not only improved hypomagnesemia but also almost completely restored cardiac and mitochondrial functions. Therefore, proactive Mg supplementation in pathological conditions induced by Mg deficiency or for those at risk of developing hypomagnesemia may suppress the development and exacerbation of certain disease states.
Assuntos
Doenças Cardiovasculares/etiologia , Hipóxia/etiologia , Deficiência de Magnésio/complicações , Mitocôndrias Cardíacas , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Animais , Pressão Sanguínea , Doenças Cardiovasculares/prevenção & controle , Doença Crônica , Suplementos Nutricionais , Modelos Animais de Doenças , Frequência Cardíaca , Magnésio/administração & dosagem , Deficiência de Magnésio/patologia , Deficiência de Magnésio/fisiopatologia , Deficiência de Magnésio/terapia , Masculino , Mitocôndrias Cardíacas/fisiologia , Membranas Mitocondriais/patologia , Ratos Sprague-Dawley , Função Ventricular EsquerdaRESUMO
Objective: The present study compared students' CBT scores during the 2-year period before ("without COVID-19") and 2-year period during ("with COVID-19") the COVID-19 pandemic, and analyzed factors associated with poor results. Materials: A total of 530 students (368 males and 162 females), who had taken CBT within the period between 2018 and 2021. Methods: Analysis was performed based on the questionnaire results, and the students' performance was compared between "without/with COVID-19" to identify the causes of poor CBT scores. Results: The overall mean IRT score was 515.5±85.4. The without and with COVID-19 groups' scores were 495.7±85.9 and 534.4±80.8, respectively (p<0.01). Among all students, 43 (8.1%) had IRT scores lower than 400 as poor CBT scores; 27 (10.4%) without and 16 (5.9%) with COVID-19, revealing a decrease in the latter. The multivariate analysis of the risk of students having poor CBT scores showed that students with poor performance during the third year (odds ratio:7.02), starting preparation for CBT late (2.19), and not taking any practice examination (4.58) are more likely to have poor CBT results. Conclusions: Due to the COVID-19 pandemic, students spent more time on online home study, and this may have consequently improved their CBT scores. Such learning performance is desirable for medical students, but they have lost the opportunity to gain valuable experiences that they could have acquired through extracurricular activities, such as club activities. In this respect, we cannot simply be pleased by the improvement in students' CBT scores.
RESUMO
Objectives: This study assessed the effects of oral porcine placental extract (PPE) on sleep quality of healthy volunteers not satisfied with their sleep. Design: This study used a randomized, placebo-controlled, double-blind, cross-over clinical pilot study. Setting: This study was conducted under an outpatient multicenter setting in Japan. Interventions: A total of 20 healthy Japanese volunteers aged between 28 and 73, whose Pittsburgh Sleep Quality Index global scores were between 6 and 10, successfully completed the study. At first, PPE at 300 mg/kg or placebo was ingested for 2 weeks. Then, after a 2-week washout period, each group ingested under a cross-over setting the opposite sample (placebo or PPE) for another 2 weeks. Main Outcome Measures: Objective measurement of the sleep made with an activity tracker and subjective measurements of sleep quality by use of St. Mary's Hospital Sleep Questionnaire were done just before and after the administration time slots. Results: No effect of PPE on the sleep length was observed. Several measures in the subjective St. Mary's Hospital Sleep Questionnaire, i.e., changes in Q5 (sleep depth) and Q9 (sleep wellness) between pre- and post-ingestions, were significantly different between groups in the direction of improvement of subjective sleep quality in the PPE group. Conclusions: Although oral PPE at 300 mg/day for 2 weeks did not affect the length of sleep itself, it significantly improved several measures of subjective sleep quality. These results suggest that PPE might be a way to improve sleep quality without hypnotic drugs. Clinical Trial Registration: www.umin.ac.jp/ctr/, identifier: UMIN000026468.
RESUMO
In atherosclerosis progression, atherosclerotic plaques develop upon accumulated foam cells derived from macrophages that take up modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Lipopolysaccharide (LPS) exacerbates atherosclerosis by enhancing the expression of scavenger receptors and thus increasing the uptake of modified LDL into macrophages. However, the signaling pathways that mediate LPS and scavenger receptor expression have not been fully elucidated. We used mouse bone marrow-derived macrophages and investigated the effects of LPS in vitro. LPS enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-1 (STAT-1). Inhibitors of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) pathway (U0126 and PD0325901) suppressed the uptake of acetylated-LDL (Ac-LDL) and the expression of CD204 but not CD36 in LPS-activated macrophages. Inhibitors of the Janus tyrosine kinase (JAK)-STAT pathway (ruxolitinib and tofacitinib) suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. We next injected LPS into the peritoneal cavity of mice and analyzed the effects of LPS. MEK inhibitor U0126 suppressed the uptake of Ac-LDL and the expression of CD204 but not CD36 in LPS-activated macrophages. JAK inhibitor ruxolitinib suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. These results suggest that scavenger receptors in LPS-activated mouse macrophages are regulated through a JAK-STAT-dependent pathway. Although further evaluation is necessary, JAK-STAT inhibition could be useful in atherosclerosis therapy, at least for atherosclerosis exacerbated by LPS.
Assuntos
Janus Quinases/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Receptores Depuradores/metabolismo , Fatores de Transcrição STAT/metabolismo , Animais , Antígenos CD36/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Receptores Depuradores Classe A/metabolismoRESUMO
INTRODUCTION: The involvement of serotonin (5-HT) in increased lower urinary tract symptoms in aging is unclear. We sought to compare voiding function and 5-HT induced detrusor contraction between young and aged rats. METHODS: This study used young (2- to 3-month-old) and aged (26- to 30-month-old) male Fischer 344 rats. 1. Rats were housed in individual metabolic cages, and then the total volume of urination, volume per micturition, voiding frequency, and voiding interval were analyzed. 2. Using urinary bladder body strips, developed tension was recorded after cumulative addition of 5-HT (1-100â¯nM) in the absence or presence of tetrodotoxin (1⯵M), and in the presence of tetrodotoxin with ketanserin (0.3-3⯵M) or naftopidil (1 and 3⯵M). We examined the effects of atropine, ketanserin, and naftopidil on electrical field stimulation (EFS)-induced contraction. RESULTS: 1. Compared to young rats, aged rats exhibited decreased voiding frequency and increased volume per micturition, but total volume of urination (normalized to body weight) did not differ. Moreover, voiding interval was significantly prolonged in aged rats during the active period. 2. In the presence of tetrodotoxin, pEC50 of 5-HT were significantly lower in aged rats than in young rats (Pâ¯<â¯0.01), but the maximal response to 5-HT was not altered in the aged bladder. Ketanserin inhibited 5-HT-induced contraction in both groups, while suppression by naftopidil was relatively limited, especially in aged rats. EFS induced neurogenic contraction in a frequency-dependent manner. Atropine-resistant contraction was not inhibited by naftopidil, but was potentiated by ketanserin. CONCLUSIONS: Urination intervals were extended in aged rats, indicating that urination rhythm changed. In the senescent rat bladder, 5-HT induced detrusor contraction, but the effect of 5-HT and the naftopidil-sensitive contractile force were weaker than those in young rats. Additionally, 5-HT did not contribute to the increase in atropine-resistant EFS-induced contractions in aged rats.
Assuntos
Contração Muscular/efeitos dos fármacos , Naftalenos/farmacologia , Piperazinas/farmacologia , Serotonina/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Atropina/farmacologia , Ketanserina/farmacologia , Masculino , Ratos , Ratos Endogâmicos F344 , Receptores 5-HT2 de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , TetrodotoxinaRESUMO
We have previously reported that increased extracellular and intracellular Ca2+ lead to adipocyte accumulation in bone marrow stromal cells (BMSCs). However, strategies to suppress high Ca2+-enhanced adipocyte accumulation have not been reported. We examined the effects of the diacylglycerol analog phorbol 12-myristate 13-acetate (PMA) on proliferation and adipogenesis of mouse primary BMSCs. We used 9 mM CaCl2 and 100 nM ionomycin to increase extracellular Ca2+ and intracellular Ca2+, respectively. PMA suppressed the expression of both C/EBPα and PPARγ under normal adipogenesis, adipogenesis + CaCl2, and adipogenesis + ionomycin conditions. PMA enhanced proliferation under normal adipogenesis conditions but suppressed proliferation under adipogenesis + CaCl2 and adipogenesis + ionomycin conditions. PMA did not affect the accumulation of adipocytes under normal adipogenesis conditions but suppressed adipocyte accumulation under adipogenesis + CaCl2 and adipogenesis + ionomycin conditions. These results suggest that the PMA-dependent pathway is an important signaling pathway to suppress high Ca2+-enhanced adipocyte accumulation.
Assuntos
Adipogenia/efeitos dos fármacos , Cálcio/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ionomicina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Only a few bone marrow-derived macrophages (BM-MΦ) are positive for macrophage colony-stimulating factor receptor (M-CSFR). Thus, a method is needed to increase the proportion of BM-MΦ that are positive for M-CSFR to facilitate the investigation of the effects of M-CSFR downregulation on various diseases. We used mouse primary BM-MΦ to evaluate the expression of M-CSFR on the cytoplasmic membrane using flow cytometry. Treatment with a reducing agent, dithiothreitol (DTT), increased the proportion of BM-MΦ that were positive for M-CSFR, and this increase was time dependent. We next determined whether DTT-treated BM-MΦ can lead to the downregulation of M-CSFR. Treatment with lipopolysaccharide (LPS) for 24 h. decreased the proportion of DTT-treated BM-MΦ that were positive for M-CSFR. These results suggest that DTT treatment increases the proportion of BM-MΦ that are positive for M-CSFR and that the upregulation of M-CSFR on BM-MΦ can be abrogated by treatment with LPS. Here, we propose a simple method to increase the number of M-CSFR-positive BM-MΦ using the reducing agent DTT, which could be useful in investigations of the relationship between the downregulation of M-CSFR and some diseases. â¢The proportion of BM-MΦ that expresses M-CSFR on the membrane increases by approximately twice following DTT treatment.
RESUMO
The Langendorff Perfused Heart Model is an experimental procedure developed at the end of the nineteenth century by Oskar Langendorff. In this procedure, an excised heart has a cannula inserted into its aorta so that the heart can be retrogradely perfused via the coronary artery. The procedure has been improved in recent times, and these improvements are used to evaluate the direct effect of medication on the heart as well as the effect of ischemia-reperfusion injury on heart function. In this chapter, we describe protocols for evaluating heart function in Langendorff perfused rat heart.
Assuntos
Coração/fisiologia , Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Perfusão/métodos , Animais , Cateterismo Cardíaco/instrumentação , Cateterismo Cardíaco/métodos , Modelos Animais de Doenças , Dissecação/métodos , Desenho de Equipamento , Frequência Cardíaca , Perfusão/instrumentação , RatosRESUMO
BACKGROUND AND AIMS: Lipopolysaccharide (LPS) is a main component of the Gram-negative bacterial cell wall and is associated with a greater risk of atherosclerosis development in periodontal disease. LPS has been reported to increase both CD36 and CD204 expression and enhance the uptake of modified low-density lipoprotein (LDL). However, the signaling pathways by which LPS enhances these expression levels and function have not been fully elucidated, although the clarification of these signaling pathways is important for identifying therapeutic targets for atherosclerosis. METHODS AND RESULTS: We have shown here that LPS activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, increased both CD204 and CD36 expression, and enhanced the uptake of acetylated-LDL (Ac-LDL) in mouse bone marrow macrophages. The MAPK/ERK kinase (MEK) inhibitors, U0126 (1 µM) and PD0325901 (10 nM), did not affect the expression of either CD36 or CD204 or the uptake of Ac-LDL under normal conditions (no treatment with LPS). In contrast, U0126 (1 µM) and PD0325901 (10 nM) blocked the LPS-induced increase in Ac-LDL uptake and CD204 expression but not CD36 expression. CONCLUSIONS: These results suggest that LPS may increase Ac-LDL uptake and enhance CD204 expression through MAPK/ERK activation and CD36 expression through an ERK-independent pathway. Since MEK inhibitors block CD204 expression in mouse BM macrophages only under LPS treatment but not under normal conditions, a MEK inhibitor might be a good candidate compound for the treatment of LPS-induced atherosclerosis.
Assuntos
Aterosclerose/induzido quimicamente , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores Depuradores Classe A/metabolismo , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Aterosclerose/imunologia , Antígenos CD36/metabolismo , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Lipoproteínas LDL/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca2+]o enhances adipocyte accumulation. We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca2+ and Sr2+) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca2+]o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1µM forskolin and 200nM IBMX) and a cAMP analog (10µM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca2+]o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca2+]o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25-40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.
Assuntos
Adipócitos/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , AMP Cíclico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptores Acoplados a Proteínas G/genética , 1-Metil-3-Isobutilxantina/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Fatores Etários , Animais , Compostos Azo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica , Insulina/farmacologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Cultura Primária de Células , Receptores de Detecção de Cálcio , Receptores Acoplados a Proteínas G/metabolismo , Coloração e Rotulagem/métodosRESUMO
In this study, we investigated the effects of a short-term and long-term high-fat diet (HFD) on morphological and functional features of fast-twitch skeletal muscle. Male C57BL/6J mice were fed a HFD (60% fat) for 4 weeks (4-week HFD) or 12 weeks (12-week HFD). Subsequently, the fast-twitch extensor digitorum longus muscle was isolated, and the composition of muscle fiber type, expression levels of proteins involved in muscle contraction, and force production on electrical stimulation were analyzed. The 12-week HFD, but not the 4-week HFD, resulted in a decreased muscle tetanic force on 100 Hz stimulation compared with control (5.1 ± 1.4 N/g in the 12-week HFD vs. 7.5 ± 1.7 N/g in the control group; P < 0.05), whereas muscle weight and cross-sectional area were not altered after both HFD protocols. Morphological analysis indicated that the percentage of type IIx myosin heavy chain fibers, mitochondrial oxidative enzyme activity, and intramyocellular lipid levels increased in the 12-week HFD group, but not in the 4-week HFD group, compared with controls (P < 0.05). No changes in the expression levels of calcium handling-related proteins and myofibrillar proteins (myosin heavy chain and actin) were detected in the HFD models, whereas fast-troponin T-protein expression was decreased in the 12-week HFD group, but not in the 4-week HFD group (P < 0.05). These findings indicate that a long-term HFD, but not a short-term HFD, impairs contractile force in fast-twitch muscle fibers. Given that skeletal muscle strength largely depends on muscle fiber type, the impaired muscle contractile force by a HFD might result from morphological changes of fiber type composition.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Contração Muscular , Fibras Musculares de Contração Rápida/fisiologia , Animais , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Cadeias Pesadas de Miosina/metabolismoRESUMO
Perlecan is a major heparan sulfate proteoglycan found in the subendothelial extracellular matrix of the vascular wall. The aim of this study was to investigate the role of perlecan in the regulation of vascular tone. A previously developed conditional perlecan-deficient mouse model was used to measure changes in the isometric force of isolated aortic rings. The vessels were first precontracted with phenylephrine, and then treated with increasing concentrations of vasorelaxants. Endothelium-dependent relaxation, elicited by acetylcholine, was significantly reduced in the perlecan-deficient aortas, whereas endothelium-independent relaxation caused by the exogenous nitric oxide donor sodium nitroprusside remained well preserved. The expression of the endothelial nitric oxide synthase (eNOS) gene, detected by real-time polymerase chain reaction, was significantly decreased in the perlecan-deficient aortas. The expression of eNOS protein detected using Western blotting was also significantly decreased in the perlecan-deficient aortas. We examined the role of perlecan in eNOS gene expression by creating perlecan knockdown human aortic endothelial cells using small interfering RNA (siRNA) for perlecan. Perlecan gene expression was significantly reduced in the perlecan siRNA-treated cells, resulting in a significant decrease in eNOS gene expression. Perlecan deficiency induced endothelial dysfunction, as indicated by a reduction in endothelium-dependent relaxation due, at least partly, to a reduction in eNOS expression. These findings suggest that perlecan plays a role in the activation of eNOS gene expression during normal growth processes.
RESUMO
Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca(2+) ([Ca(2+)]o and [Ca(2+)]i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca(2+)]o and high [Ca(2+)]i enhanced adipocyte accumulation, which suggested that increases in [Ca(2+)]o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca(2+)]o and high [Ca(2+)]i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca(2+)]i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca(2+)]o (addition of CaCl2) leads to increases in [Ca(2+)]i. Flow cytometric methods revealed that high [Ca(2+)]o suppressed the phosphorylation of ERK independently of intracellular Ca(2+). The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca(2+) provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca(2+), which results in BMSC proliferation.
Assuntos
Adipócitos/citologia , Adipogenia , Cálcio/metabolismo , Células-Tronco Mesenquimais/citologia , Adipócitos/metabolismo , Animais , Proliferação de Células , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Pulmonary arterial hypertension (PAH) is a debilitating and deadly disease with no known cure. Heart failure is a major comorbidity and a common cause of the premature death of patients with PAH. Increased asymmetrical right ventricular hypertrophy and septal wall thickening compress the left ventricular cavity and elicit diastolic heart failure. In this study, we used the Sugen5416/hypoxia/normoxia-induced PAH rat to determine whether altered pyridine nucleotide signaling in the failing heart contributes to 1) increased oxidative stress, 2) changes in metabolic phenotype, 3) autophagy, and 4) the PAH-induced failure. We found that increased reactive oxygen species, metabolic maladaptation, and autophagy contributed to the pathogenesis of right ventricular remodeling and hypertrophy that lead to left ventricular diastolic dysfunction. In addition, arterial elastance increased in PAH rats. Glucose-6-phosphate dehydrogenase is a major source of pyridine molecule (nicotinamide adenine dinucleotide phosphate), which is a substrate for nicotinamide adenine dinucleotide phosphate oxidases in the heart. Dehydroepiandrosterone, a 17-ketosteroid that reduces pulmonary hypertension and right ventricular hypertrophy, inhibited glucose-6-phosphate dehydrogenase, decreased oxidative stress, increased glucose oxidation and acetyl-coA, and reduced autophagy in the hearts of PAH rats. It also decreased arterial stiffness and improved left ventricular diastolic function. These findings demonstrate that pyridine nucleotide signaling, at least partly, mediates PAH-induced diastolic heart failure, and that reduction of glucose-6-phosphate dehydrogenase-derived nicotinamide adenine dinucleotide phosphate is beneficial to improve left ventricle diastolic function.
Assuntos
Autofagia , Insuficiência Cardíaca Diastólica/etiologia , Hipertensão Pulmonar/complicações , Hipertrofia Ventricular Direita/etiologia , Miocárdio/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca Diastólica/metabolismo , Insuficiência Cardíaca Diastólica/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Masculino , Miocárdio/metabolismo , NADPH Oxidases/metabolismo , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda , Remodelação VentricularRESUMO
DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure.
Assuntos
DNA/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Conformação de Ácido Nucleico , DNA/química , DNA/metabolismo , Nanoestruturas/químicaRESUMO
BACKGROUND: The right gastroepiploic artery is useful as an in situ arterial graft for coronary artery bypass grafting. However, the gastroepiploic artery is more likely to cause vasospasms compared with the internal thoracic artery. We hypothesized that the cause of the spasms is the stimulation of the periarterial sympathetic nerve, because the gastroepiploic artery is classified as a muscular artery. In this study, we examined whether the spasm is reduced by removing the periarterial sympathetic nerve. METHODS: Unused parts of the gastroepiploic artery were obtained from patients who underwent coronary artery bypass grafting. The vessel was cut into 2 segments, and they were assigned to control (N+) and denervation (N-) groups. The periarterial nerve was microscopically removed from the vessels of the N- group. The vessels in both groups were investigated by hematoxylin-eosin or immunohistochemical staining, and they were stimulated by electrical field stimulation with serial frequency for isometric tension measurement. RESULTS: Histologic analyses revealed that periarterial connective tissues including neuropeptide Y were removed to expose the external elastic membrane in the N- vessel, whereas they were preserved in N+. The mean contraction by electrical field stimulation with serial frequency was consistently lower in N- than in N+ (P < .05 at 20 and 50 Hz; n = 8 each). Endothelium-dependent relaxation and contractile function of the smooth muscle were similar in both groups. CONCLUSIONS: The removal of the periarterial sympathetic nerve from the human gastroepiploic artery reduced vascular contraction, elicited by peripheral nerve stimulation, without disturbing endothelial and smooth muscle contractile functions. This reduction may contribute to the prevention of vasospasms.
Assuntos
Fibras Adrenérgicas , Arteriopatias Oclusivas/prevenção & controle , Artéria Gastroepiploica/inervação , Artéria Gastroepiploica/cirurgia , Simpatectomia , Vasoconstrição , Fibras Adrenérgicas/química , Arteriopatias Oclusivas/fisiopatologia , Biomarcadores/análise , Constrição Patológica , Estimulação Elétrica , Artéria Gastroepiploica/fisiopatologia , Humanos , Contração Isométrica , Neuropeptídeo Y/análise , VasodilataçãoRESUMO
We fabricated a microfluidic device for the entrapment of nonadherent cells. Solution exchange was easily performed by introducing the solution into the cell-trapping microchannel. Immunostaining and labeling of the cell membrane of THP-1 cells were demonstrated using this device, which does not require cumbersome repetition of centrifugation and resuspension steps.
Assuntos
Membrana Celular/metabolismo , Separação Celular/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Actinas/metabolismo , Adesão Celular , Linhagem Celular , Separação Celular/instrumentação , Corantes Fluorescentes , Humanos , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismoRESUMO
We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 µM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 µM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.