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1.
Drug Metab Dispos ; 44(8): 1390-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27208383

RESUMO

Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes.


Assuntos
Azóis/farmacologia , Simulação por Computador , Indutores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2B6/biossíntese , Indutores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/biossíntese , Hepatócitos/efeitos dos fármacos , Modelos Estatísticos , Adulto , Azóis/química , Azóis/toxicidade , Células Cultivadas , Citocromo P-450 CYP2B6/genética , Indutores do Citocromo P-450 CYP2B6/química , Indutores do Citocromo P-450 CYP2B6/toxicidade , Citocromo P-450 CYP3A/genética , Indutores do Citocromo P-450 CYP3A/química , Indutores do Citocromo P-450 CYP3A/toxicidade , Interações Medicamentosas , Hepatócitos/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Análise de Componente Principal , RNA Mensageiro/biossíntese , Análise de Regressão , Relação Estrutura-Atividade , Fatores de Tempo
2.
Pharm Res ; 32(2): 604-16, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25163980

RESUMO

PURPOSE: To evaluate the impact of intestinal first-pass metabolism (Fg) by cytochrome P4503A (CYP3A) and uridine 5'-diphosphate-glucuronosyltransferases (UGT) on in vivo oral absorption of their substrate drugs. METHODS: CYP3A and UGT substrates were orally administered to portal-vein cannulated (PV) rats to evaluate their intestinal availability (Fa · Fg). In the case of CYP3A substrates, vehicle or 1-aminobenzotriazole (ABT), a potent inhibitor of CYP enzymes, was pretreated to assess Fg separately from Fa (Enzyme-inhibition method). On the other hand, since potent inhibitors of UGT have not been identified, Fg of UGT substrate was calculated from total amount of metabolites generated in enterocytes (Metabolite-distribution method). RESULTS: After oral administration of CYP3A substrates in ABT-pretreated rats, the portal and systemic plasma concentrations of the metabolite were nearly the same, indicating almost complete inhibition of intestinal CYP3A-mediated metabolism. Using Enzyme-inhibition method, Fg of midazolam (1 mg/kg) was calculated as 0.71. Additionally, total amount of raloxifene-6-glucuronide generated in enterocytes after oral administration of raloxifene was estimated using Metabolite-distribution method and Fg of raloxifene (0.98 µmol/kg) was calculated as 0.21. CONCLUSIONS: PV rats enabled in vivo quantitative assessment of intestinal first-pass metabolism by CYP3A and UGT. This method is useful for clarifying the cause of low bioavailability.


Assuntos
Absorção Intestinal/fisiologia , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismo , Veia Porta/metabolismo , Administração Oral , Animais , Cateterismo/métodos , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Inibidores do Citocromo P-450 CYP3A/metabolismo , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Veia Porta/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia
3.
Drug Metab Dispos ; 41(8): 1514-21, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23686319

RESUMO

The purpose of this study was to evaluate the impact of intestinal efflux transporters on the in vivo oral absorption process. Three model drugs-fexofenadine (FEX), sulfasalazine (SASP), and topotecan (TPT)-were selected as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and P-gp and BCRP substrates, respectively. The drugs were orally administered to portal vein-cannulated rats after pretreatment with zosuquidar (ZSQ), P-gp inhibitor, and/or Ko143, BCRP inhibitor. Intestinal availability (Fa·Fg) of the drugs was calculated from the difference between portal and systemic plasma concentrations. When rats were orally pretreated with ZSQ, Fa·Fg of FEX increased 4-fold and systemic clearance decreased to 75% of the control. In contrast, intravenous pretreatment with ZSQ did not affect Fa·Fg of FEX, although systemic clearance decreased significantly. These data clearly show that the method presented herein using portal vein-cannulated rats can evaluate the effects of intestinal transporters on Fa·Fg of drugs independently of variable systemic clearance. In addition, it was revealed that 71% of FEX taken up into enterocytes underwent selective efflux via P-gp to the apical surface, while 79% of SASP was effluxed by Bcrp. In the case of TPT, both transporters were involved in its oral absorption. Quantitative analysis indicated a 3.5-fold higher contribution from Bcrp than P-gp. In conclusion, the use of portal vein-cannulated rats enabled the assessment of the impact of efflux transporters on intestinal absorption of model drugs. This experimental system is useful for clarifying the cause of low bioavailability of various drugs.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Absorção , Adenosina/análogos & derivados , Adenosina/farmacologia , Administração Oral , Animais , Cateterismo , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas , Compostos Heterocíclicos de 4 ou mais Anéis , Masculino , Veia Porta , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfassalazina/farmacocinética , Terfenadina/análogos & derivados , Terfenadina/farmacocinética , Topotecan/farmacocinética
4.
Biochem Biophys Res Commun ; 410(3): 608-13, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21684256

RESUMO

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Corpos Embrioides/citologia , Pâncreas Exócrino/citologia , Ativinas/farmacologia , Amilases/biossíntese , Carboxipeptidases A/biossíntese , Quimotripsina/biossíntese , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/enzimologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Fator 3-beta Nuclear de Hepatócito/biossíntese , Humanos , Lipase/biossíntese , Niacinamida/farmacologia , Pâncreas Exócrino/enzimologia , Elastase Pancreática/biossíntese , Fatores de Transcrição SOXF/biossíntese , Tretinoína/farmacologia
5.
Clin Transl Immunology ; 10(5): e1282, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33976880

RESUMO

OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed piggyBac-modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a piggyBac-based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term in vitro and in vivo anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term in vitro settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116+ R/R AML.

6.
Retrovirology ; 6: 43, 2009 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-19435492

RESUMO

BACKGROUND: In vivo studies of HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Although transgenic rats that express the HIV-1 receptor complex hCD4 and hCCR5 are susceptible to infection, HIV-1 replicates very poorly in these animals. To demonstrate the molecular basis for developing a better rat model for HIV-1 infection, we evaluated the effect of human CyclinT1 (hCycT1) and CRM1 (hCRM1) on Gag p24 production in rat T cells and macrophages using both established cell lines and primary cells prepared from hCycT1/hCRM1 transgenic rats. RESULTS: Expression of hCycT1 augmented Gag production 20-50 fold in rat T cells, but had little effect in macrophages. Expression of hCRM1 enhanced Gag production 10-15 fold in macrophages, but only marginally in T cells. Expression of both factors synergistically enhanced p24 production to levels approximately 10-40% of those detected in human cells. R5 viruses produced in rat T cells and macrophages were fully infectious. CONCLUSION: The expression of both hCycT1 and hCRM1 appears to be fundamental to developing a rat model that supports robust propagation of HIV-1.


Assuntos
Ciclinas/biossíntese , HIV-1/fisiologia , Carioferinas/biossíntese , Macrófagos/virologia , Receptores Citoplasmáticos e Nucleares/biossíntese , Linfócitos T/virologia , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Ciclina T , Modelos Animais de Doenças , Proteína do Núcleo p24 do HIV/biossíntese , Humanos , Ratos , Ratos Transgênicos , Proteína Exportina 1
7.
Nihon Yakurigaku Zasshi ; 153(4): 186-191, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30971659

RESUMO

Drug metabolism in the liver is a major factor affecting pharmacokinetics of drugs, and cytochrome P450s (P450s) are major enzymes responsible for it. Since drug-drug interactions (DDIs) can affect the pharmacokinetics of concomitantly administrated drugs, it may limit the drug therapy such as dose adjustment and contraindications for co-administration and lead to dose adjustment and contraindications for co-administration. DDI is thus one of the risk factors to be reduced in the lead-optimization stage. Therefore, it is important to estimate DDI risk in the early drug discovery stage and develop candidates with low DDI risk. P450 induction is one of the important mechanisms causing DDIs and the activation of nuclear receptors is involved in this phenomenon. In this manuscript, the mechanism and evaluation methods of P450 induction are briefly reviewed, and then the new in silico methods for the prediction of P450 induction, which have been recently established by us, and its application to drug development are introduced.


Assuntos
Descoberta de Drogas , Simulação por Computador , Sistema Enzimático do Citocromo P-450 , Interações Medicamentosas , Preparações Farmacêuticas
8.
Retrovirology ; 5: 90, 2008 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-18840303

RESUMO

BACKGROUND: Human T cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Immunological studies have suggested that insufficient host T cell response to HTLV-I is a potential risk factor for ATL. To understand the relationship between host T cell response and HTLV-I pathogenesis in a rat model system, we have developed an activation and detection system of HTLV-I Tax-specific cytotoxic T lymphocytes (CTLs) by Epitope expressing Single-Chain Trimers (SCTs) of MHC class I. RESULTS: We have established expression vectors which encode SCTs of rat MHC-I (RT1.Al) with Tax180-188 peptide. Human cell lines transfected with the established expression vectors were able to induce IFN-gamma and TNF-alpha production by a Tax180-188-specific CTL line, 4O1/C8. We have further fused the C-terminus of SCTs to EGFP and established cells expressing SCT-EGFP fusion protein on the surface. By co-cultivating the cells with 4O1/C8, we have confirmed that the epitope-specific CTLs acquired SCT-EGFP fusion proteins and that these EGFP-possessed CTLs were detectable by flow cytometric analysis. CONCLUSION: We have generated a SCT of rat MHC-I linked to Tax epitope peptide, which can be applicable for the induction of Tax-specific CTLs in rat model systems of HTLV-I infection. We have also established a detection system of Tax-specific CTLs by using cells expressing SCTs fused with EGFP. These systems will be useful tools in understanding the role of HTLV-I specific CTLs in HTLV-I pathogenesis.


Assuntos
Epitopos de Linfócito T/genética , Expressão Gênica , Produtos do Gene tax/genética , Genes MHC Classe I , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Linfócitos T Citotóxicos/imunologia , Ativação Transcricional , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Produtos do Gene tax/imunologia , Vetores Genéticos/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/virologia , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/virologia
9.
J Pharm Sci ; 107(9): 2479-2488, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29802934

RESUMO

CYP2C enzymes play key roles in drug metabolism, and clinical drug-drug interactions caused by CYP2C induction have been reported. The aim of this study was to establish a method to predict the potency of CYP2C inductions considering the mechanism. We first investigated the relations of CYP2C induction with CYP3A4 or CYP2B6 induction in human hepatocytes after 48-h exposure with 19 inducers. The fold-induction values of CYP2C8 and CYP2C9 were well correlated with those of CYP3A4, whereas the inducers were separated into 2 groups showing different correlations with CYP2B6 induction for CYP2C8 and CYP2C9 induction. In the regression models established, the fold-induction values of CYP2C8 and CYP2C9 were well expressed as the functions of those of CYP3A4 and CYP2B6, while no such obvious correlation was observed for CYP2C19 induction. These results suggest that CYP2Cs are not simply coinduced with CYP3A4 and that CYP2C8 and CYP2C9 inductions are regulated by both pregnane X receptor and constitutive androstane receptor with different contributions. Finally, simple correlations were proposed using the experimental Emax values obtained and plasma concentrations of CYP2C9 substrates from the literature, and positive correlations were observed. These data provide methods to estimate the clinical impact of CYP2C9 induction from in vitro data.


Assuntos
Criopreservação , Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/enzimologia , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adulto , Idoso , Receptor Constitutivo de Androstano , Relação Dose-Resposta a Droga , Previsões , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas
10.
Int J Alzheimers Dis ; 2017: 8584205, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29333315

RESUMO

Presenilin 1 and presenilin 2 (PS1 and PS2) play a critical role in γ-secretase-mediated cleavage of amyloid-ß precursor protein (APP) and the subsequent generation of ß-amyloid peptides. The purpose of the present study was to test whether PS2 mutation accelerates the onset of contextual fear memory deficits in a mouse model of AD that expresses a mutation (K670N/M671L) of the human APP with the Swedish mutation (Tg2576 mice). In the present study, an APP/PS2 double-transgenic mouse model (PS2Tg2576) was generated by crossbreeding transgenic mice carrying the human mutant PS2 (N141I) with Tg2576 mice. Contextual fear conditioning was tested in PS2Tg2576 mice aged 3, 4, 6, and 10-12 months. PS2Tg2576 mice showed a tendency of lower freezing behavior as early as 3 months of age, but significant memory impairment was observed from the age of 4 months. The cognitive impairment was more prominent at ages of 6 and 10-12 months. In contrast, Tg2576 mice aged 3 and 4 months exhibited successful acquisition of contextual fear learning, but Tg2576 mice aged 6 months or older showed significantly impaired fear memory. These results show that PS2 mutation significantly accelerates the onset of fear memory deficits in the APP AD model mice.

11.
World J Stem Cells ; 7(5): 883-93, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26131319

RESUMO

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 10(6)) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm(2) including 2.1 × 10(6) cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaOH maceration.

12.
J Med Chem ; 57(23): 9844-54, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25383422

RESUMO

The discovery of a new series of potent phosphodiesterase 7 (PDE7) inhibitors is described. Novel thieno[3,2-d]pyrimidin-4(3H)-one hit compounds were identified from our chemical library. Preliminary modifications of the hit compounds were performed, resulting in the discovery of a fragment-sized compound (10) with highly improved ligand efficiency. Compound design was guided by structure-activity relationships and computational modeling. The 6-substituted derivatives of the thienopyrimidinone showed diminished activity and enzyme selectivity. However, synthesis of the 7-substituted derivatives resulted in the discovery of 28e, a desirable lead compound that selectively inhibits PDE7 with single-digit nanomolar potency while displaying potent cellular efficacy.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Inibidores de Fosfodiesterase/síntese química , Pirimidinonas/síntese química , Animais , Simulação por Computador , Humanos , Camundongos , Inibidores de Fosfodiesterase/farmacologia , Pirimidinonas/farmacologia , Relação Estrutura-Atividade
13.
Ann Anat ; 192(5): 314-21, 2010 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-20801009

RESUMO

Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications.


Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Conexina 43/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Junções Comunicantes/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Células Estromais/fisiologia , Regulação para Cima
14.
Arch Pharm (Weinheim) ; 341(9): 548-53, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18618491

RESUMO

Non-invasive measurement and visualization of free radicals in vivo would be important to clarify their roles in the pathogenesis of free radical-associated diseases. Nitroxyl radicals can react with free radicals and be derivatized to achieve specific cellular/subcellular localizing capabilities while retaining the simple spectral features useful in imaging. Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity via the Overhauser Effect. In this study, we synthesized various nitroxyl probes having (15)N nuclei and deuterium, and measured the enhancement factor for Overhauser-enhanced magnetic resonance imaging experiments. (15)N-D-4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl ((15)N-D-oxo-TEMPO) has the highest enhancement factor compared with other nitroxyl probes. The proton signal enhancement was higher for (15)N-labeled nitroxyl probes when compared to the (14)N-labeled analogues because of the reduced spectral multiplicity of the I=1/2 nucleus. Furthermore, this enhancement is proportional to the line width and number of electron spin resonance lines of nitroxyl radicals. Finally, we compared the Overhauser-enhanced magnetic resonance image of (15)N-labeled, deuterated 4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl with that of (14)N-H-TEMPOL. These results suggested that the selective deuteration of the nitroxyl probes enhanced the signal-to-noise ratio and thereby improved spatial and temporal resolutions.


Assuntos
Imageamento por Ressonância Magnética/métodos , Óxidos de Nitrogênio/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Indicadores e Reagentes , Imagens de Fantasmas
15.
Proc Natl Acad Sci U S A ; 103(5): 1463-8, 2006 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-16432234

RESUMO

MRI has provided significant clinical utility in the diagnosis of diseases and will become a powerful tool to assess phenotypic changes in genetically engineered animals. Overhauser enhanced MRI (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity by means of the Overhauser effect. Several studies have demonstrated noninvasive assessment of reactive oxygen species generation in small animals by using low frequency electron spin resonance (ESR) spectroscopy/imaging and nitroxyl radicals. In vivo ESR signal intensities of nitroxyl radicals decrease with time after injection; and the decreases are enhanced by reactive oxygen species, generated in oxidative disease models in a site-specific manner. In this study, we show images of nitroxyl radicals with different isotopes by changing the external magnetic field for ESR irradiation between (14)N and (15)N nuclei in field-cycled OMRI. OMRI simultaneously obtained dual images of two individual chemical processes. Oxidation and reduction were monitored in a rate-dependent manner at nanometer scale by labeling membrane-permeable and -impermeable nitroxyl radicals with (14)N and (15)N nuclei. Phantom objects containing ascorbic acid-encapsulated liposomes with membrane-permeable radicals but not membrane-impermeable ones show a time-dependent decrease of the OMRI image intensity. The pharmacokinetics in mice was assessed with OMRI after radical administration. This OMRI technique with dual probes should offer significant applicability to nanometer scale molecular imaging and simultaneous assessment of independent processes in gene-modified animals. Thus, it may become a powerful tool to clarify mechanisms of disease and to monitor pharmaceutical therapy.


Assuntos
Imageamento por Ressonância Magnética/métodos , Óxidos de Nitrogênio , Oxirredução , Animais , Membrana Celular/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Processamento de Imagem Assistida por Computador , Lipossomos/química , Masculino , Camundongos , Modelos Genéticos , Nanotecnologia , Oxigênio/química , Oxigênio/metabolismo , Imagens de Fantasmas , Marcadores de Spin , Fatores de Tempo
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