Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Biol Chem ; 282(46): 33507-33514, 2007 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-17827152

RESUMO

We previously reported that CD31(bright) cells, which were sorted from cultured AC133(+) cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31(+) cells or CD31(-) cells, suggesting that CD31(bright) cells may be endothelial precursor cells. In this study, we found that CD31(bright) cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133(+) cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31(bright) cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.


Assuntos
Células Endoteliais/citologia , Trombopoetina/fisiologia , Antígenos CD34/biossíntese , Linhagem Celular , Separação Celular , Citocinas/metabolismo , Citometria de Fluxo/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Magnetismo , Modelos Biológicos , Óxido Nítrico Sintase Tipo III/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Estrutura Terciária de Proteína , Transdução de Sinais , Trombopoetina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
J Virol Methods ; 143(1): 95-103, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17433454

RESUMO

To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV.


Assuntos
Hepacivirus/isolamento & purificação , Vírus da Hepatite A Humana/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Separação Imunomagnética/métodos , Complexo Antígeno-Anticorpo , Hepacivirus/imunologia , Vírus da Hepatite A Humana/imunologia , Vírus da Hepatite B/imunologia , Humanos , Microesferas , Polietilenoimina , Reação em Cadeia da Polimerase
3.
J Cell Physiol ; 211(1): 189-96, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17133348

RESUMO

Previously, we suggested that the phosphatidylinositol 3-kinase (PI3K)-p70 S6 kinase (p70 S6K) pathway plays an important role in granulocyte colony-stimulating factor (G-CSF)-dependent enhancement of the neutrophilic differentiation and proliferation of HL-60 cells. While atypical protein kinase C (PKC) has been reported to be a regulator of p70 S6K, abundant expression of PKCiota was observed in myeloid and lymphoid cells. Therefore, we analyzed the participation of PKCiota in G-CSF-dependent proliferation. The maximum stimulation of PKCiota was observed from 15 to 30 min after the addition of G-CSF. From 5 to 15 min into this lag time, PKCiota was found to translocate from the nucleus to the membrane. At 30 min it re-translocated to the cytosol. This dynamic translocation of PKCiota was also observed in G-CSF-stimulated myeloperoxidase-positive cells differentiated from cord blood cells. Small interfering RNA for PKCiota inhibited G-CSF-induced proliferation and the promotion of neutrophilic differentiation of HL-60 cells. These data indicate that the G-CSF-induced dynamic translocation and activation processes of PKCiota are important to neutrophilic proliferation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Isoenzimas/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Proteína Quinase C/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Células HL-60 , Humanos , Isoenzimas/genética , Células Jurkat , Células K562 , Neutrófilos/citologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/genética , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Fatores de Tempo
4.
J Steroid Biochem Mol Biol ; 94(4): 303-9, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15857749

RESUMO

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.


Assuntos
Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Receptores X de Retinoides/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Alitretinoína , Antígeno CD11b/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Histona Acetiltransferases , Nitroazul de Tetrazólio , Coativador 1 de Receptor Nuclear , Receptores de Formil Peptídeo/metabolismo , Receptores X de Retinoides/agonistas , Receptores X de Retinoides/metabolismo , Ressonância de Plasmônio de Superfície , Tretinoína
5.
Biochem Pharmacol ; 66(1): 133-40, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12818373

RESUMO

We have previously suggested that phosphatidylinositol 3-kinase (PI3K)/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R(+)) and -negative (Trf-R(-)) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in differentiation and proliferation of both cell types, with a particular focus on c-Myc. Similar to p70 S6K, we found that the expression of c-Myc in Trf-R(+) cells is also higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, indicating that the extent of c-Myc inhibition by these inhibitors correlates with a reduction in proliferation, and that c-Myc is downstream from PI3K/p70 S6K. We also determined phosphorylation of the 4E-binding protein 1 (4E-BP1), which is regulated downstream of the mammalian target of rapamycin. The addition of G-CSF failed to enhance the phosphorylation state of 4E-BP1 of HL-60 cells 2 days after DMSO differentiation. An antisense oligonucleotide for c-myc inhibited both G-CSF-dependent enhancement of c-Myc expression and proliferation in Trf-R(+) cells, but did not enhance the differentiation in terms of O(2)(-)-generating ability or fMLP-R expression. In contrast, antisense oligonucleotide for c-myc promoted fMLP-R on non-treated HL-60 cells. We therefore conclude that the PI3K/p70 S6K/c-Myc cascade plays an important role in neutrophilic proliferation in HL-60 cells. Unlike that of rapamycin, however, the antisense oligonucleotide for c-myc could not promote differentiation of Trf-R(+) cells cultured with G-CSF, indicating that another target downstream of p70 S6K may control the differentiation of HL-60 cells in terms of the signal transduction of G-CSF.


Assuntos
Diferenciação Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Neutrófilos/citologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Androstadienos/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Imunossupressores/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Sirolimo/farmacologia , Wortmanina
6.
J Cell Physiol ; 195(1): 119-29, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12599215

RESUMO

To clarify the process of endothelial differentiation, we isolated AC133(+) cells and induced the in vitro differentiation of these cells into endothelial cells. AC133(+) cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin-coated dishes in the presence of vascular endothelial growth factor. Time-course analysis of the alteration of endothelial markers on cultured AC133(+) cells revealed that the expression of CD31 (PECAM-1) on AC133(+) cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133(+) cells. CD31-bright cells, which were sorted from cultured AC133(+) cells, differentiated more efficiently into endothelial cells than had CD31-positive or CD31-negative cells, suggesting that CD31-bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31(+) cells derived from cultured AC133(+) cells that are able to differentiate to endothelial cells as precursor cells.


Assuntos
Endotélio Vascular/citologia , Glicoproteínas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Células-Tronco/citologia , Células-Tronco/metabolismo , Antígeno AC133 , Antígenos CD , Antígenos de Diferenciação/biossíntese , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Separação Celular , Células Cultivadas , Fatores de Crescimento Endotelial/farmacologia , Fibronectinas/farmacologia , Citometria de Fluxo , Humanos , Separação Imunomagnética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Leucócitos Mononucleares/citologia , Linfocinas/farmacologia , Peptídeos , Células-Tronco/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
7.
Biochem Biophys Res Commun ; 300(3): 770-4, 2003 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-12507517

RESUMO

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.


Assuntos
Anexina A3/análise , Anexina A3/biossíntese , Hepatócitos/química , Hepatócitos/metabolismo , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Hepatócitos/classificação , Masculino , Peso Molecular , Mapeamento de Peptídeos , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
J Biochem ; 134(6): 827-34, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14769871

RESUMO

The involvement of protein phosphatases in the activation of superoxide (O2-)- generating enzyme in human neutrophils was examined using calyculin A, an inhibitor of protein phosphatase type 1 and 2A. Calyculin A inhibited the phorbol myristate acetate (PMA)- and opsonized zymosan (OZ)-activated O2- generation by human neutrophils. This inhibitory effect of calyculin A on PMA-activated O2- generation was reversed by the addition of KT5926, a specific inhibitor of myosin light chain kinase and Ca2+/calmodulin-dependent protein kinase II. These results suggest that the addition of calyculin A may cause hyperphosphorylation of some protein(s) that plays a crucial role in the PMA-dependent activation of O2- generating enzyme, and that this protein hyperphosphorylation may be evoked by a KT5926-sensitive kinase or its downstream kinase. Whereas two-dimensional analysis involving 32P revealed that calyculin A caused the hyperphosphorylation of many proteins, KT5926 mainly reduced the calyculin A-induced hyperphosphorylation of a 67 kDa protein in activated neutrophils, suggesting that the hyperphosphorylation of the 67 kDa protein might inhibit the PMA-dependent activation of NADPH oxidase. The 67 kDa cytosolic protein was moderately phosphorylated on the addition of PMA. On the other hand, in the absence of calyculin A, KT5926 inhibited both PMA-induced O2- generation and phosphorylation of the 67 kDa protein. Amino acid sequence analysis of peptides derived from the 67 kDa protein revealed that the 67 kDa protein was identical to L-plastin, an actin-bundling protein. We conclude that optimally phosphorylated L-plastin may play some crucial role in the activation of NADPH oxidase.


Assuntos
NADPH Oxidases/metabolismo , Fosfoproteínas/metabolismo , Superóxidos/metabolismo , Sequência de Aminoácidos , Carbazóis/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Toxinas Marinhas , Glicoproteínas de Membrana , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oxazóis/antagonistas & inibidores , Oxazóis/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Fosforilação/efeitos dos fármacos , Superóxidos/antagonistas & inibidores
9.
Arch Biochem Biophys ; 405(1): 21-31, 2002 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12176053

RESUMO

Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.


Assuntos
Neutrófilos/citologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Androstadienos/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Movimento Celular , Separação Celular , Dimetil Sulfóxido/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células HL-60 , Humanos , Immunoblotting , Neutrófilos/metabolismo , Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Testes de Precipitina , Receptores da Transferrina/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Tretinoína/farmacologia , Wortmanina
10.
Biologicals ; 30(1): 69-76, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11846431

RESUMO

This study was undertaken to establish a standard method for determination of the activity of human (h) thrombomodulin (TM). The reactions mainly consisted of formation of a h-TM and h-thrombin complex, activation of h-protein C by the complex and digestion of substrate by activated h-protein C. Linear time-dependent formation of p -nitroaniline from the substrate, S-2366, was observed up to 12 min during measurement of the activity of urinary h-TM (uh-TM) reference material by the standard method. Therefore, 10 minutes was established as the reaction time in the standard method. In the standard method, we defined the activity of h-TM forming 0.1 micromol of p -nitroaniline per min in the reaction as 1 JRS Unit. Recombinant h-TM (rh-TM) and uh-TM reference material gave rectilinear dose-response curves within a certain range of specific activities by their original methods in the standard method. The validity of the standard method was assessed based on the coefficients of variation (CV) obtained in the various measurements of h-TM. Intra-batch precision (CV) of h-thrombin and h-protein C was 2.90% and 6.57%, respectively, in the measurement of uh-TM activity. The intra-sample, inter-day, and inter-laboratory precision (CV) was 1.30%, 1.63% and 5.02%, respectively, in the measurement of the first Japanese reference standard for h-TM coded TJRS1. In these assays, the activity of the first Japanese standard was also determined and found to be 205 JRS units per ampoule. When the stability of the Japanese reference standard was assessed by measuring of the standard stored under various thermal conditions, the predicted loss of activity assuming monomolecular degradation according to the Arrhenius equation was less than 3.0% during 103 years at -20 degrees C. These results indicate that the standard method is appropriate for determination of the activity of h-TM and that the Japanese reference standard for h-TM, whose activity was determined here, can be stored at -20 degrees C for long periods without loss of activity.


Assuntos
Padrões de Referência , Trombomodulina/análise , Análise de Variância , Compostos de Anilina/química , Química Clínica/normas , Relação Dose-Resposta a Droga , Fibrinolíticos/química , Temperatura Alta , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Japão , Laboratórios/normas , Peptídeos/farmacologia , Temperatura , Trombina/análise , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...