RESUMO
BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening disease, resulting in refractory heart failure. An immune disorder underlies the pathophysiology associated with heart failure progression. Invariant natural killer T (iNKT) cell activation is a prospective therapeutic strategy for ischemic heart disease. However, its efficacy in nonischemic cardiomyopathy, such as DCM, remains to be elucidated, and the feasible modality for iNKT cell activation in humans is yet to be validated. METHODS: Dendritic cells isolated from human volunteers were pulsed with α-galactosylceramide ex vivo, which were used as α-galactosylceramide-pulsed dendritic cells (αGCDCs). We treated DCM mice harboring mutated troponin TΔK210/ΔK210 with αGCDCs and evaluated the efficacy of iNKT cell activation on heart failure in DCM mice. Furthermore, we investigated the molecular basis underlying its therapeutic effects in these mice and analyzed primary cardiac cells under iNKT cell-secreted cytokines. RESULTS: The number of iNKT cells in the spleens of DCM mice was reduced compared with that in wild-type mice, whereas αGCDC treatment activated iNKT cells, prolonged survival of DCM mice, and prevented decline in the left ventricular ejection fraction for 4 weeks, accompanied by suppressed interstitial fibrosis. Mechanistically, αGCDC treatment suppressed TGF (transforming growth factor)-ß signaling and expression of fibrotic genes and restored vasculature that was impaired in DCM hearts by upregulating angiopoietin 1 (Angpt1) expression. Consistently, IFNγ (interferon gamma) suppressed TGF-ß-induced Smad2/3 signaling and the expression of fibrotic genes in cardiac fibroblasts and upregulated Angpt1 expression in cardiomyocytes via Stat1. CONCLUSIONS: Immunomodulatory cell therapy with αGCDCs is a novel therapeutic strategy for heart failure in DCM.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Camundongos , Humanos , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/terapia , Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/metabolismo , Volume Sistólico , Função Ventricular Esquerda , Fibrose , Células Dendríticas/metabolismo , Terapia Baseada em Transplante de Células e TecidosRESUMO
The phosphorous supply crisis is a major challenge for a sustainable society, and the algal industry is not unrelated to this crisis. Recycling phosphorus from sewage wastewater is a potential way to address this issue. We previously developed amorphous calcium silicate hydrates (aCSH) as excellent phosphorus recovery materials. In this study, we designed a phosphorus recovery process using aCSH in a pilot-scale facility connected to a sewage wastewater treatment plant, and demonstrated the production of microalgal biomass using phosphorous-containing aCSH (P_aCSH). As a result, high phosphorous recovery rates (>80%) were obtained throughout the year. The carbohydrate-rich microalga Pseudoneochloris sp. NKY372003 was cultivable with P_aCSH. The biomass and carbohydrate productivity of this microalga with P_aCSH was comparable to that with conventional media. Approximately 94% of the phosphorus in P_aCSH was recycled into the biomass. This study successfully demonstrated the recycling the phosphorus recovered from wastewater for microalgal cultivation by aCSH.
Assuntos
Fósforo , Águas Residuárias , Biomassa , Compostos de Cálcio , SilicatosRESUMO
Thrombin increases the cytosolic Ca(2+) concentrations and induces NO production by activating proteinase-activated receptor 1 (PAR(1)) in vascular endothelial cells. The store-operated Ca(2+) influx is a major Ca(2+) influx pathway in non-excitable cells including endothelial cells and it has been reported to play a role in the thrombin-induced Ca(2+) signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca(2+) content, thereby regulating the store-operated Ca(2+) influx. However, the functional role of STIM1 in the thrombin-induced Ca(2+) influx and NO production in endothelial cells still remains to be elucidated. Fura-2 and diaminorhodamine-4M fluorometry was utilized to evaluate the thrombin-induced changes in cytosolic Ca(2+) concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1-targeted siRNA suppressed the STIM1 expression and the thapsigargin-induced Ca(2+) influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca(2+) influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca(2+) influx and a partial reduction of the NO production induced by thrombin. The thrombin-induced Ca(2+) influx exhibited the similar sensitivity toward the Ca(2+) influx inhibitors to that seen with the thapsigargin-induced Ca(2+) influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR(1)-mediated Ca(2+) influx and Ca(2+)-dependent component of the NO production in endothelial cells.
Assuntos
Sinalização do Cálcio , Endotélio Vascular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Óxido Nítrico/biossíntese , Receptor PAR-1/metabolismo , Animais , Aorta/citologia , Sequência de Bases , Bloqueadores dos Canais de Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Células Cultivadas , Células Endoteliais , Endotélio Vascular/citologia , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/metabolismo , RNA Interferente Pequeno , Receptor PAR-1/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Alinhamento de Sequência , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Suínos , Trombina/metabolismo , Fatores de TempoRESUMO
Laboratory-scale acidophilic nitrifying sequencing-batch reactors (ANSBRs) were constructed by seeding with sewage-activated sludge and cultivating with ammonium-containing acidic mineral medium (pH 4.0) with or without a trace amount of yeast extract. In every batch cycle, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate. Attempts to detect nitrifying functional genes in the fully acclimated ANSBRs by PCR with previously designed primers mostly gave negative results. 16S rRNA gene-targeted PCR and a subsequent denaturating gradient gel electrophoresis analysis revealed that a marked change occurred in the bacterial community during the overall period of operation, in which members of the candidate phylum TM7 and the class Gammaproteobacteria became predominant at the fully acclimated stage. This result was fully supported by a 16S rRNA gene clone library analysis, as the major phylogenetic groups of clones detected (>5% of the total) were TM7 (33%), Gammaproteobacteria (37%), Actinobacteria (10%), and Alphaproteobacteria (8%). Fluorescence in situ hybridization with specific probes also demonstrated the prevalence of TM7 bacteria and Gammaproteobacteria. These results suggest that previously unknown nitrifying microorganisms may play a major role in ANSBRs; however, the ecophysiological significance of the TM7 bacteria predominating in this process remains unclear.
Assuntos
Bactérias/classificação , Reatores Biológicos/microbiologia , Biota , Meios de Cultura/química , Esgotos/microbiologia , Compostos de Amônio/metabolismo , Bactérias/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Nitrificação , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Using a 3D-imaging-create-function server and network services by IP-VPN, we began to deliver 3D images to the remote institution. An indication trial of the primary image, a rotary trial of a 3D image, and a reproducibility trial were studied in order to examine the practicality of using the system in a real network between Hakodate and Sapporo (communication distance of about 150 km). In these trials, basic data (time and receiving data volume) were measured for every variation of QF (quality factor) or monitor resolution. Analyzing the results of the system using a 3D image delivery server of our hospital with variations in the setting of QF and monitor resolutions, we concluded that this system has practicality in the remote interpretation-of-radiogram work, even if the access point of the region has a line speed of 6 Mbps.
Assuntos
Imageamento Tridimensional/métodos , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Consulta Remota/métodos , Reprodutibilidade dos Testes , SoftwareRESUMO
Proteinase-activated receptors 1 and 4 (PAR(1) and PAR(4)) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca(2+) requirement for the NO production between PAR(1) and PAR(4). The activation of PAR(1) by the activating peptide (PAR(1)-AP) elevated cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR(4) activation by PAR(4)-AP consistently induced NO production without an appreciable [Ca(2+)](i) elevation in three types of endothelial cells. The PAR(1)-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR(4)-mediated NO production was resistant. NO production following the PAR(1) and PAR(4) activation was significantly inhibited by pertussis toxin, but it was resistant to a Galpha(q/11) inhibitor, YM254890 [(1R)-1-[(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl]-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR(1)-mediated Ca(2+) signal. PAR(4)-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR(1)-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR(4) stimulation. In conclusion, PAR(1) and PAR(4) engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR(4) preferably activates Galpha(i/o) and induced NO production in a manner mostly independent of Ca(2+) but dependent on the PI3K/Akt pathway, whereas PAR(1) activates both the Ca(2+)-dependent and -independent mechanisms.
Assuntos
Cálcio/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Androstadienos/farmacologia , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Morfolinas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Toxina Pertussis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Suínos , WortmaninaRESUMO
Acute systemic hypoxia causes significant increases in human skeletal muscle sympathetic nerve activity (MSNA), heart rate and ventilation. This phenomenon is thought to be primarily mediated by excitation of peripheral chemoreceptors sensing a fall in arterial free oxygen partial pressure (Pa,O2). We directly tested the role of Pa,O2 on MSNA (peroneal microneurography), heart rate, ventilation and leg haemodynamics (n = 7-8) at rest and during rhythmic handgrip exercise by using carbon monoxide (CO) to mimic the effect of systemic hypoxia on arterial oxyhaemoglobin (approximately 20 % lower O2Hba), while normalising or increasing Pa,O2 (range 40-620 mmHg). The four experimental conditions were: (1) normoxia (Pa,O2 approximately 110 mmHg; carboxyhaemoglobin (COHb) approximately 2 %); (2) hypoxia (Pa,O2 approximately 40 mmHg; COHb approximately 2 %); (3) CO + normoxia (Pa,O2 approximately 110 mmHg; COHb approximately 23 %); and (4) CO + hyperoxia (Pa,O2 approximately 620 mmHg; COHb ~24 %). Acute hypoxia augmented sympathetic burst frequency, integrated MSNA, heart rate and ventilation compared to normoxia over the entire protocol (7-13 bursts min-1, 100-118 %, 13-17 beats min-1, 2-4 l min-1, respectively, P < 0.05). The major new findings were: (1) CO + normoxia and CO + hyperoxia also elevated MSNA compared to normoxia (63-144 % increase in integrated MSNA; P < 0.05) but they did not increase heart rate (62-67 beats min-1) or ventilation (6.5-6.8 l min-1), and (2) despite the 4-fold elevation in MSNA with hypoxaemia and exercise, resting leg blood flow, vascular conductance and O2 uptake remained unchanged. In conclusion, the present results suggest that increases in MSNA with CO are not mediated by activation of the chemoreflex, whereas hypoxia-induced tachycardia and hyperventilation are mediated by activation of the chemoreflex in response to the decline in Pa,O2. Our findings also suggest that Pa,O2 is not an obligatory signal involved in the enhanced MSNA with reduced blood oxygenation.
Assuntos
Exercício Físico/fisiologia , Frequência Cardíaca/fisiologia , Hipóxia/fisiopatologia , Perna (Membro)/irrigação sanguínea , Músculo Esquelético/fisiologia , Oxigênio/sangue , Sistema Nervoso Simpático/fisiologia , Adulto , Pressão Sanguínea/fisiologia , Monóxido de Carbono , Força da Mão/fisiologia , Humanos , Masculino , Músculo Esquelético/inervação , Oxiemoglobinas/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Mecânica Respiratória/fisiologia , Descanso/fisiologia , Decúbito Dorsal/fisiologiaRESUMO
A family of specific carrier protein designated as monocarboxylate transporter (MCT) has been known to transport the lactate and other moncarboxylates in mammalian cells. We hypothesized the presence of serum protein in human circulation that may works as a lactate carrier and that biochemical structure would possesses common structure with MCT on the plasma membrane. Immunoblot analysis with an anti-MCT1 polyclonal antibody suggested the presence of a 44-kDa protein in human circulation and N-terminal amino acid sequencing exhibited a stretch of 14 amino acids which is completely identical to MCT1. The unbound fractions from the GST-MCTI fusion protein-immobilized glutathione sepharose 4B column demonstrated that lactic acid concentration began to increase with one fraction delay compared to Sepharose 4B and GST-immobilized column. When lactic acid was washed away with PBS, lactic acid concentrations in the effuluent constantly decreased in both Sepharose 4B and GST-immobilized column. However, GST-MCT1-immobilized column showed specific convex curve from fraction approximately 3 mM of lactate and demonstrated wash out delay compared to Sepharose 4B and GST-immobilized column. These observations demonstrated biochemical and immunological similarities between a 44-kDa protein purified from human serum and MCT1 present on the plasma membrane. The studies on MCT1-fusion protein suggested possible functional properties of a 44-kDa protein as a lactate buffer by holding and unhand a lactate according to the lactate concentration in human blood. The experiments described herein have suggested the existence of lactate carrier in human circulation which is free from plasma membrane.
Assuntos
Animais , Membrana Celular/metabolismo , Cricetinae , DNA Complementar/metabolismo , Eritrócitos/metabolismo , Biblioteca Gênica , Glutationa Transferase/metabolismo , Ventrículos do Coração/patologia , Humanos , Immunoblotting , Ácido Láctico/metabolismo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: It has been suspected for some time that patients with chronic heart failure (CHF) have abnormal muscle metabolism, so in the present study the muscle metabolism of the arm and leg were compared by (31)P magnetic resonance spectroscopy ((31)P-MRS) to examine the relationship to exercise tolerance. METHODS AND RESULTS: The study group comprised 13 patients and 11 normal controls. Calf-plantar and forearm-wrist flexion were performed to evaluate the metabolic capacity assessed as the phosphocreatine breakdown rate (PCr-slope) and muscle pH at a submaximal (70% peak) work rate (submax-pH). Exercise of both the arm and leg resulted in an earlier decrease in PCr and muscle pH in patients with CHF compared with controls. There were significant correlations between peak oxygen uptake (peak V(O2)) and the PCr-slope in both limbs in patients with CHF (forearm: r=0.63, p<0.05; calf: r=0.60, p<0.05), but no correlations in normal controls. There was a close correlation between the ventilatory anaerobic threshold (AT) and the PCr-slope in the calf (r=0.85, p<0.01), but not in the forearm in patients with CHF. Submax-pH in both upper and lower limbs was not significantly correlated to peak V(O2) or AT in either patients with CHF or controls. CONCLUSIONS: Although metabolic abnormalities during exercise are seen in both arms and legs, leg muscle abnormalities, in particular, are closely related to systemic exercise intolerance in patients with CHF.