RESUMO
Methodical screening of safe and efficient drug candidate compounds is crucial for drug development. A high-throughput and accurate compound evaluation method targeting the central nervous system can be developed using in vitro neural networks. In particular, an evaluation system based on a human-derived neural network that can act as an alternative to animal experiments is desirable to avoid interspecific differences. A microelectrode array (MEA) is one such evaluation system, and can measure in vitro neural activity; however, studies on compound evaluation criteria and in vitro to in vivo extrapolation are scarce. In this study, we identified the parameters that can eliminate the effects of solvents from neural activity data obtained using MEA allow for accurate compound evaluation. Additionally, we resolved the issue associated with compound evaluation criteria during MEA using principal component analysis by considering the neuronal activity exceeding standard deviation (SD) of the solvent as indicator of seizurogenic potential. Overall, 10 seizurogenic compounds and three negative controls were assessed using MEA-based co-cultured human-induced pluripotent stem cell-derived neurons and astrocytes, and primary rat cortical neurons. In addition, we determined rat cerebrospinal fluid (CSF) concentrations during tremor and convulsion in response to exposure to test compounds. To characterize the in vitro to in vivo extrapolation and species differences, we compared the concentrations at which neuronal activity exceeding the SD range of the solvent was detectable using the MEA system and rat CSF concentration.
Assuntos
Astrócitos , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Ratos , Neurônios , Convulsões , SolventesRESUMO
The gyromagnetic factor of the low-lying E=251.96(9) keV isomeric state of the nucleus ^{99}Zr was measured using the time-dependent perturbed angular distribution technique. This level is assigned a spin and parity of J^{π}=7/2^{+}, with a half-life of T_{1/2}=336(5) ns. The isomer was produced and spin aligned via the abrasion-fission of a ^{238}U primary beam at RIKEN RIBF. A magnetic moment |µ|=2.31(14)µ_{N} was deduced showing that this isomer is not single particle in nature. A comparison of the experimental values with interacting boson-fermion model IBFM-1 results shows that this state is strongly mixed with a main νd_{5/2} composition. Furthermore, it was found that monopole single-particle evolution changes significantly with the appearance of collective modes, likely due to type-II shell evolution.
RESUMO
Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is mainly expressed on S1 and S2 segment of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is increased under diabetic conditions, selective inhibition of SGLT2 is a potential therapeutic target for the treatment of diabetes. We have recently shown that an inhibitor of SGLT2 has anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing advanced glycation end products formation and oxidative stress generation in the kidney. However, the direct effects of SGLT2 inhibitor on tubular cell damage remain unclear. In this study, we investigated the effects of tofogliflozin, a highly selective inhibitor of SGLT2 on oxidative stress generation, inflammatory and proapoptotic reactions in cultured human proximal tubular cells exposed to high glucose. Tofogliflozin dose-dependently suppressed glucose entry into tubular cells. High glucose exposure (30 mM) for 4 and 24 h significantly increased oxidative stress generation in tubular cells, which were suppressed by the treatment of tofogliflozin or an antioxidant N-acetylcysteine (NAC). Monocyte chemoattractant protein-1 (MCP-1) gene expression and apoptotic cell death were induced by 4 h- and 8 day-exposure to high glucose, respectively, both of which were also blocked by tofogliflozin or NAC. The present study suggests that SGLT2-mediated glucose entry into tubular cells could stimulate oxidative stress and evoke inflammatory and proapoptotic reactions in this cell type. Blockade of glucose reabsorption in tubular cells by SGLT2 inhibitor might exert beneficial effects on tubulointerstitial damage in diabetic nephropathy.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Benzidrílicos/uso terapêutico , Glucose/farmacologia , Glucosídeos/uso terapêutico , Inflamação/tratamento farmacológico , Túbulos Renais Proximais/patologia , Estresse Oxidativo/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose , Apoptose/genética , Compostos Benzidrílicos/farmacologia , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Humanos , Inflamação/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transportador 2 de Glucose-Sódio/metabolismoRESUMO
Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.
Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/prevenção & controle , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/farmacologia , Serpinas/metabolismo , Animais , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/etiologia , Humanos , Rim/metabolismo , Masculino , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
We have investigated the excitation intensity dependence of the singlet fission in a crystalline rubrene by means of femtosecond transient absorption microspectroscopy. When a rubrene microcrystal was excited to higher energy levels than that of the lowest singlet excited (S1) state with a 397 nm femtosecond laser pulse, a triplet excited state was formed through two pathways of the singlet fission, i.e. the direct fission from higher vibrational levels of the S1 state with a time constant of 2.2 ps and the thermally activated fission from the S1 state in a few tens of ps. The time constant of the thermally activated fission changed from 35 to 17 ps for increasing of the laser fluence from 0.65 to 18 mJ cm-2 per pulse, although that of the direct fission was constant with the excitation laser intensity. On the other hand, the yield of the triplet formation was independent of the intensity. We also examined the temperature dependence of the singlet fission and demonstrated the activation energy of the thermally activated fission to be 0.21 eV. Based on the experimental results, we considered the excitation intensity dependence of the singlet fission of the rubrene crystal in terms of the effect of transient local heating on a ps time scale after femtosecond laser excitation owing to the nonradiative vibrational relaxation from the higher vibrational level to the lower one in the S1 state.
RESUMO
Advanced glycation endproducts (AGEs) and their receptor (RAGE) play a role in vascular complications in diabetes. We have previously shown that 17ß-estradiol at 10 nmol/l, a nearly identical plasma concentration to that during mid-pregnancy, up-regulates RAGE expression in endothelial cells. The finding might suggest the involvement of 17ß-estradiol in the deterioration of vascular complications in diabetes during pregnancy. However, the effects of the selective estrogen receptor modulator, bazedoxifene, on oxidative and inflammatory reactions in AGEs-exposed endothelial cells remain unknown. In this study, we addressed the issue. Ten nmol/l 17ß-estradiol increased RAGE and monocyte chemoattractant protein-1 (MCP-1) gene and protein expression in human umbilical vein endothelial cells (HUVECs), both of which were blocked by 10 nmol/l bazedoxifene. Bazedoxifene at 10 nmol/l also significantly inhibited the AGEs-induced superoxide generation, RAGE and MCP-1 gene and protein expression in HUVECs. The present study suggests that blockade of the AGEs-RAGE axis by bazedoxifene might be a novel therapeutic target for preventing vascular damage in diabetes, especially in postmenopausal women.
Assuntos
Quimiocina CCL2/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Indóis/administração & dosagem , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Superóxidos/química , Células Cultivadas , Quimiocina CCL2/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/genéticaRESUMO
Advanced glycation end products (AGEs) and their receptor (RAGE) system are involved in diabetic nephropathy. Angiotensin-converting enzyme 2 (ACE 2) plays a protective role against cardiovascular and renal injury by stimulating the production of angiotensin-(1-7) [Ang-(1-7)], an antagonist of angiotensin II (Ang II). However, effects of the AGEs-RAGE axis on ACE 2 expression in mesangial cells remain unknown. We examined here the role of ACE 2 in the AGEs-RAGE-induced mesangial cell damage and investigated whether olmesartan, one of the Ang II type 1 receptor blockers (ARB), prevented the deleterious effects of AGEs via restoration of ACE 2 and Ang-(1-7) level. AGEs significantly increased superoxide generation, upregulated RAGE mRNA level, and decreased ACE 2 gene expression and Ang-(1-7) production in mesangial cells, all of which were blocked by olmesartan, but not by a different type of ARB, azilsartan. An antioxidant, N-acetylcysteine or RAGE-antibodies also restored the decrease in ACE 2 mRNA level in AGEs-exposed mesangial cells. Moreover, olmesartan, but not azilsartan completely inhibited the AGEs-induced increase in vascular cell adhesion molecule-1 (VCAM-1) mRNA level in mesangial cells, which was abolished by the treatment with A-779, an antagonist of Ang-(1-7) receptor, Mas receptor. Our present study suggests that olmesartan could block the AGEs-induced VCAM-1 gene induction in mesangial cells by restoring the downregulated ACE 2 levels and subsequently stimulating the Ang-(1-7)-Mas receptor axis. Restoration of ACE 2 levels and blockade of renin-angiotensin system by olmesartan might be a promising strategy for the treatment of diabetic nephropathy.
Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Imidazóis/farmacologia , Células Mesangiais/metabolismo , Peptidil Dipeptidase A/metabolismo , Tetrazóis/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Angiotensina I , Enzima de Conversão de Angiotensina 2 , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/enzimologia , Fragmentos de Peptídeos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Superóxidos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Metformin use has been reported to decrease breast cancer incidence and mortality in diabetic patients. We have previously shown that advanced glycation end products (AGEs) and their receptor (RAGE) interaction stimulate growth and/or migration of pancreatic cancer and melanoma cells. However, effects of metformin on AGEs-RAGE axis in breast cancers remain unknown. We examined here whether and how metformin could block the AGEs-induced growth and vascular endothelial growth factor (VEGF) expression in MCF-7 breast cancer cells. Cell proliferation was measured with an electron coupling reagent WST-1 based colorimetric assay. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. AGEs significantly increased cell proliferation of MCF-7 cells, which was completely prevented by the treatment with 0.01 or 0.1 mM metformin or anti-RAGE antibodies. Furthermore, metformin at 0.01 mM completely suppressed the AGEs-induced upregulation of RAGE and VEGF mRNA levels in MCF-7 cells. An inhibitor of AMP-activated protein kinase, compound C significantly blocked the growth-inhibitory and RAGE and VEGF suppressing effects of metformin in AGEs-exposed MCF-7 cells. Our present study suggests that metformin could inhibit the AGEs-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing RAGE gene expression via AMP-activated protein kinase pathway. Metformin may protect against breast cancer expansion in diabetic patients by blocking the AGEs-RAGE axis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Produtos Finais de Glicação Avançada/farmacologia , Metformina/farmacologia , Receptores Imunológicos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Ichthyosis bullosa of Siemens (IBS) is an autosomal dominant skin disorder that resembles epidermolytic hyperkeratosis (EHK). We have identified mutations in two families originally diagnosed with EHK and in four families diagnosed with IBS at the same codon in the highly conserved carboxy terminal of the rod domain of keratin 2e, thus revealing a mutational hot spot. Our results allow a differential diagnosis to be made between IBS and EHK at the genetic level and we suggest that patients diagnosed with EHK, but lacking keratin K1 or K10 mutations, should be re-examined for mutations in their K2e genes.
Assuntos
Hiperceratose Epidermolítica/genética , Ictiose/genética , Queratinas/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Primers do DNA/genética , Diagnóstico Diferencial , Feminino , Genes Dominantes , Humanos , Hiperceratose Epidermolítica/diagnóstico , Hiperceratose Epidermolítica/patologia , Ictiose/diagnóstico , Ictiose/patologia , Queratina-2 , Queratinas/química , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Mutação , LinhagemRESUMO
Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in tubulointerstitial damage in diabetic nephropathy. Recently, metformin has been shown to ameliorate tubular injury both in cell culture and diabetic animal model. However, effects of metformin on AGEs-induced tubular cell apoptosis and damage remain unknown. We examined here whether and how metformin could block the AGEs-RAGE-elicited tubular cell injury in vitro. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. Reactive oxygen species (ROS) generation was measured with dihydroethidium staining. Apoptosis was evaluated by DNA fragmentation and annexin V expression level. AGEs upregulated RAGE mRNA levels and subsequently increased ROS generation and intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and transforming growth factor-ß gene expression in human renal proximal tubular cells, all of which were significantly blocked by the treatment of 0.01 and 0.1 mM metformin. Compound C, an inhibitor of AMP-activated protein kinase significantly blocked the effects of metformin on RAGE gene expression and ROS generation in AGEs-exposed tubular cells. Furthermore, metformin dose-dependently inhibited the AGEs-induced apoptotic cell death of tubular cells; 1 mM metformin completely suppressed the pro-apoptotic effects of AGEs in 2 different assay systems. Our present study suggests that metformin could inhibit the AGEs-induced apoptosis and inflammatory and fibrotic reactions in tubular cells probably by reducing ROS generation via suppression of RAGE expression through AMP-activated protein kinase activation. Metformin may protect against tubular cell injury in diabetic nephropathy by blocking the AGEs-RAGE-ROS axis.
Assuntos
Produtos Finais de Glicação Avançada/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Metformina/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Nefropatias Diabéticas/prevenção & controle , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Hipoglicemiantes/antagonistas & inibidores , Hipoglicemiantes/uso terapêutico , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Metformina/antagonistas & inibidores , Metformina/uso terapêutico , Terapia de Alvo Molecular , Concentração Osmolar , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVES: The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. METHODS: Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. RESULTS: The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. CONCLUSIONS: Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology.
Assuntos
Artrite Reumatoide/genética , Quimiocinas/genética , Regulação da Expressão Gênica/fisiologia , Fator Regulador 1 de Interferon/genética , Fator de Transcrição STAT1/genética , Membrana Sinovial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Quimiocina CCL5/genética , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Quimiocinas/metabolismo , Análise por Conglomerados , Feminino , Humanos , Imuno-Histoquímica , Inflamação/genética , Masculino , Microdissecção , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/genética , Regulação para CimaRESUMO
OBJECTIVE: To investigate relationship between serum hyaluronan (HA) level and the presence and severity of radiographic knee osteoarthritis (OA) as well as degree of knee pain in Japanese population. DESIGN: A total of 616 volunteers participated in this study. Based on the Kellgren-Lawrence (K-L) grade, participants were radiographically classified into three groups: Normal (K-L grade 0 or 1), Moderate (grade 2) and Severe (grade 3 or 4). The degree of knee pain was quantified by visual analogue scale (VAS) and Knee injury and Osteoarthritis Outcome Score (KOOS) Pain. Serum HA levels were compared among the Normal, Moderate and Severe groups, and the relationship between serum HA level and the severity of knee OA was analyzed after age, sex and body mass index (BMI) were adjusted. In addition, the correlation between serum HA level and the degree of knee pain was analyzed in each group. RESULTS: Regarding relationship between serum HA level and the severity of radiographic knee OA, serum HA levels of the Moderate and Severe groups were significantly higher than in the Normal group (P<0.001). Furthermore, serum HA level correlated with the severity of radiographic knee OA (r=0.289, P<0.001) after adjusting for age, sex and BMI. Serum HA level correlated with VAS of knee pain and/or KOOS Pain in the Normal and Moderate groups. CONCLUSION: Serum HA level has the potential to be useful for the diagnosis of the presence and severity of knee OA.
Assuntos
Povo Asiático , Ácido Hialurônico/sangue , Osteoartrite do Joelho/sangue , Dor/etiologia , Idoso , Envelhecimento/sangue , Biomarcadores/sangue , Feminino , Humanos , Japão , Articulação do Joelho/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/etnologia , Dor/diagnóstico , Medição da Dor , Qualidade de Vida , Radiografia , Índice de Gravidade de DoençaRESUMO
Adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) and ABCG1 play a crucial role in macrophage cholesterol efflux, which is a novel therapeutic target for atherosclerosis. Advanced glycation end products (AGE) and their receptor RAGE axis is involved in accelerated atherosclerosis in diabetes as well. However, the role of AGE-RAGE axis in macrophage cholesterol efflux is not fully understood. We examined here whether AGE-RAGE axis could impair cholesterol efflux from human macrophage cells, THP-1 cells by suppressing ABCA1 and ABCG1 expression. We further investigated the effects of rosuvastatin on cholesterol efflux from AGE-exposed THP-1 cells. AGE increased reactive oxygen species generation in THP-1 cells, which was completely inhibited by rosuvastatin, anti-RAGE-antibody or diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase. The antioxidative effect of rosuvastatin on AGE-exposed THP-1 cells was significantly prevented by geranylgeranyl pyrophosphate (GGPP). AGE decreased ABCA1 and ABCG1 mRNA levels, and subsequently reduced cholesterol efflux from THP-1 cells, which was prevented by GGPP. DPI mimicked the effects of rosuvastain. The results demonstrated that rosuvastatin could inhibit the AGE-induced reduction of THP-1 macrophage cholesterol efflux by suppressing NADPH oxidase activity via inhibition of geranylgeranylation of Rac-1. Our present study provides a novel beneficial aspect of rosuvastatin in diabetes; rosuvastain may prevent the development and progression of atherosclerosis in diabetes by not only reducing serum cholesterol level, but also by improving cholesterol efflux from foam cells of the arterial wall via blocking the harmful effects of AGE on macrophages.
Assuntos
Colesterol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fluorbenzenos/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , Macrófagos/metabolismo , NADPH Oxidases/metabolismo , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Macrófagos/efeitos dos fármacos , NADPH Oxidases/genética , Prenilação , Rosuvastatina Cálcica , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Sitagliptin is a stable inhibitor of dipeptidyl peptidase-IV, a responsible enzyme that mainly inactivates glucagon-like peptide-1 (GLP-1), and now one of the widely used agents for the treatment of diabetes. However, effects of sitagliptin on vascular injury are largely unknown. Since advanced glycation end products (AGEs) and their receptor (RAGE) axis contribute to vascular damage in diabetes, we investigated here whether sitagliptin inhibits the AGE-RAGE-induced endothelial cell damage in vitro. Although effects of 10 pM GLP-1 or 0.5 µM sitagliptin monotherapy on RAGE gene and protein expression were modest, combination therapy completely blocked the AGE-induced increase in RAGE mRNA and protein levels in human umbilical vein endothelial cells (HUVEC). AGEs induced reactive oxygen species (ROS) generation and reduced endothelial nitric oxide synthase (eNOS) mRNA level in HUVEC, both of which were also completely blocked by the treatment with 10 pM GLP-1 and 0.5 µM sitagliptin, but not with GLP-1 or sitagliptin monotherapy. Further, anti-RAGE antibody restored the decrease in eNOS mRNA level in AGE-exposed HUVEC. The present study suggests that sitagliptin augments the effects of GLP-1 on eNOS mRNA level in AGE-exposed HUVEC by suppressing RAGE expression and subsequent ROS generation. Sitagliptin may work as a vasoprotecitve agent in diabetes by blocking the AGE-RAGE axis.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Substâncias Protetoras/farmacologia , Pirazinas/farmacologia , Receptores Imunológicos/metabolismo , Triazóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Soroalbumina Bovina/farmacologia , Fosfato de SitagliptinaRESUMO
Screening for drug discovery targeting the central nervous system requires the establishment of efficient and highly accurate toxicity test methods that can reduce costs and time while maintaining high throughput using the function of an in vitro neural network. In particular, an evaluation system using a human-derived neural network is desirable in terms of species difference. Despite the attention, the microelectrode array (MEA) is attracting among the evaluation systems that can measure in vitro neural activity, an effective analysis method for evaluation of toxicity and mechanism of action has not yet been established. Here we established analytical parameters and multivariate analysis method capable of detecting seizure liability of drugs using MEA measurement of human iPS cell-derived neurons. Using the spike time series data of all drugs, we established periodicity as a new analytical parameter. Periodicity has facilitated the detection of responses to seizurogenic drugs, previously difficult to detect with conventional analytical parameters. By constructing a multivariate analytical method that identifies a parameter set that achieves an arbitrary condition, we found that the parameter set comprising total spikes, maximum frequency (MF), inter- MF interval (IMFI), coefficient of variance of IMFI, and periodicity can uniformly detect the seizure liability of seizurogenic drugs with different mechanisms of action. Seizurogenic drugs were suggested to increase the regularity of the network burst in MEA measurements in human iPS cell-derived neurons.
Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Microeletrodos , Neurônios , Análise de Componente Principal , Convulsões/induzido quimicamenteRESUMO
Brain organoids with three-dimensional structure and tissue-like function are highly demanded for brain disease research and drug evaluation. However, to our knowledge, methods for measuring and analyzing brain organoid function have not been developed yet. This study focused on the frequency components of an obtained waveform below 500 Hz using planner microelectrode array (MEA) and evaluated the response to the convulsants pentylenetetrazol (PTZ) and strychnine as well as the antiepileptic drugs (AEDs) perampanel and phenytoin. Sudden and persistent seizure-like firing was observed with PTZ administration, displaying a concentration-dependent periodic activity with the frequency component enhanced even in one oscillation characteristic. On the other hand, in the administration of AEDs, the frequency of oscillation decreased in a concentration-dependent manner and the intensity of the frequency component in one oscillation also decreased. Interestingly, at low doses of phenytoin, a group of synchronized bursts was formed, which was different from the response to the perampanel. Frequency components contained information on cerebral organoid function, and MEA was proven useful in predicting the seizure liability of drugs and evaluating the effect of AEDs with a different mechanism of action. In addition, frequency component analysis of brain organoids using MEA is an important analysis method to perform in vitro to in vivo extrapolation in the future, which will help explore the function of the organoid itself, study human brain developments, and treat various brain diseases.
RESUMO
BACKGROUND/OBJECTIVES: Depression and hopelessness are frequently experienced in chronic kidney disease (CKD) and are generally associated with lessened physical activity. The aim of this study was to quantify the associations between sarcopenia as determined by SARC-F with both depression and hopelessness. DESIGN AND SETTING: This multicenter cohort study involving cross-sectional and longitudinal analyses was conducted in a university hospital and four general hospitals, each with a nephrology center, in Japan. PARTICIPANTS: Participants consisted of 314 CKD patients (mean age 67.6), some of whom were receiving dialysis (228, 73%). MEASUREMENTS: The main exposures were depression, measured using the Center for Epidemiologic Studies Depression (CES-D) questionnaire, and hopelessness, measured using a recently developed 18-item health-related hope scale (HR-Hope). The outcomes were sarcopenia at baseline and one year after, measured using the SARC-F questionnaire. Logistic regression models were applied. RESULTS: The cross-sectional and longitudinal analyses included 314 and 180 patients, respectively. Eighty-nine (28.3%) patients experienced sarcopenia at baseline, and 44 (24.4%) had sarcopenia at the one-year follow-up. More hopelessness (per 10-point lower, adjusted odds ratio [AOR]: 1.33, 95% confidence interval [95% CI] 1.12-1.58), depression (AOR: 1.87, 95% CI 1.003-3.49), age (per 10-year higher, AOR: 1.70, 95% CI 1.29-2.25), being female (AOR: 2.67, 95% CI 1.43-4.98), and undergoing hemodialysis (AOR, 2.92; 95% CI, 1.41-6.05) were associated with a higher likelihood of having baseline sarcopenia. More hopelessness (per 10-point lower, AOR: 1.69, 95% CI 1.14-2.51) and depression (AOR: 4.64, 95% CI: 1.33-16.2) were associated with a higher likelihood of having sarcopenia after one year. CONCLUSIONS: Among patients with different stages of CKD, both hopelessness and depression predicted sarcopenia. Provision of antidepressant therapies or goal-oriented educational programs to alleviate depression or hopelessness can be useful options to prevent sarcopenia.
Assuntos
Insuficiência Renal Crônica , Sarcopenia , Idoso , Estudos de Coortes , Estudos Transversais , Depressão/epidemiologia , Feminino , Esperança , Humanos , Masculino , Diálise Renal , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/terapia , Sarcopenia/complicações , Sarcopenia/epidemiologiaRESUMO
Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes/macrophages by means of a bacterial surface-associated protein, filamentous hemagglutinin (FHA) and the leukocyte integrin, complement receptor 3 (CR3, alpha M beta 2, CD11b/CD18). We show that an FHA Arg-Gly-Asp site induces enhanced B. pertussis binding to monocytes, and that this enhancement is blocked by antibodies directed against CR3. Enhancement requires a monocyte signal transduction complex, composed of leukocyte response integrin (alpha? beta 3) and integrin-associated protein (CD47). This complex is known to upregulate CR3 binding activity. Thus, a bacterial pathogen enhances its own attachment to host cells by coopting a host cell signaling pathway.
Assuntos
Adesinas Bacterianas , Aderência Bacteriana , Bordetella pertussis/fisiologia , Hemaglutininas/fisiologia , Antígeno de Macrófago 1/fisiologia , Monócitos/microbiologia , Transdução de Sinais , Fatores de Virulência de Bordetella , Sequência de Bases , Humanos , Dados de Sequência Molecular , Oligopeptídeos/fisiologia , Receptores de Fibronectina/fisiologiaRESUMO
The development of rod and cone photoreceptor cells was investigated in the retinas of Pacific bluefin tuna larvae and juveniles, using RET-P1 monoclonal antibody labeling to identify photoreceptors. At 60 h after hatching, which was about when feeding began, opsin (presumably green opsin (Rh2)) was expressed in the outer segments of cone cells. At 15 days after hatching (dah), although many labeled cone cells were observed in the dorsal retina, the same type of cone cells had partially appeared in the ventral retina. The presence of rod cell bodies was confirmed by the expression of Rh1 opsin at 15 dah. At 21 dah, the presence of outer segments of rod cells was confirmed by the expression of Rh1 opsin and by morphology. The observations suggest that the cone cells were substantially operable upon the development of their outer segment at around the beginning of the post-larval stage, and the rod cells began to function at around 15 to 21 dah, before and during metamorphosis.