Assuntos
Escleroderma Sistêmico , Dermatopatias , Biomarcadores , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/patologia , Esclerose/patologia , Pele/patologia , Dermatopatias/patologiaAssuntos
Eritema/patologia , Doença Relacionada a Imunoglobulina G4/patologia , Doença de Kimura/patologia , Doenças Pulmonares Intersticiais/complicações , Administração Oral , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/diagnóstico , Eosinófilos/imunologia , Eosinófilos/patologia , Eritema/diagnóstico , Evolução Fatal , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Humanos , Doença Relacionada a Imunoglobulina G4/diagnóstico , Doença Relacionada a Imunoglobulina G4/imunologia , Arcada Osseodentária/patologia , Doença de Kimura/complicações , Linfadenopatia/diagnóstico por imagem , Linfadenopatia/patologia , Linfócitos/imunologia , Linfócitos/patologia , Plasmócitos/imunologia , Plasmócitos/patologia , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Resultado do TratamentoRESUMO
LIGHT [the name of which is derived from 'homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpes simplex virus entry mediator (HVEM), and expressed by T lymphocytes'], is a member of the tumour necrosis factor superfamily that is involved in various inflammatory diseases. We aimed to estimate the relevance of plasma LIGHT levels as a biomarker for atopic dermatitis (AD). In order to understand the putative role of LIGHT in AD pathogenesis, we also investigate the effects of LIGHT on a monocytic cell line, human acute monocytic leukaemia cell line (THP-1). We examined plasma LIGHT levels, total serum IgE, serum value of CCL17 and peripheral blood eosinophil counts in patients with AD and healthy subjects. The effects of LIGHT on activation and apoptosis in THP-1 cells were also investigated. The plasma concentrations of LIGHT in AD patients were significantly higher than those in healthy individuals and the concentrations decreased as the symptoms were improved by treatment. The LIGHT plasma concentrations correlated with IgE levels and the Severity Scoring of AD (SCORAD) index. In addition, LIGHT stimulation increased expression of CD86 and induced production of interleukin-1ß in THP-1 cells. Apoptosis was inhibited, the Bcl-2 level increased and the caspase-3 level decreased in THP-1 cells stimulated with LIGHT, compared to unstimulated control cells. These results suggest that plasma LIGHT levels may be one of the promising biomarkers for AD.
Assuntos
Dermatite Atópica/sangue , Dermatite Atópica/imunologia , Eosinófilos/efeitos dos fármacos , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Adulto , Apoptose/efeitos dos fármacos , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Biomarcadores/sangue , Linhagem Celular Tumoral , Quimiocina CCL17/sangue , Progressão da Doença , Eosinófilos/patologia , Feminino , Humanos , Imunoglobulina E/sangue , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/farmacologia , Regulação para Cima , Adulto JovemRESUMO
WHAT IS KNOWN AND OBJECTIVE: Montelukast, a cysteinyl leukotriene receptor 1 antagonist, is safe and efficacious in patients with asthma. The mechanisms underlying the significant interpatient variability in response to montelukast are not clear but are believed to be, in part, because of genetic variability. METHODS: To examine the associations between polymorphisms in candidate genes in the leukotriene pathway and outcomes in patients with asthma on montelukast for 4-8 weeks, we evaluated the changes in peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV(1·0) ) and patients' subjective symptom before and after montelukast treatment. DNA was collected from 252 Japanese participants. RESULTS AND DISCUSSION: Two single-nucleotide polymorphisms (SNPs) in the ALOX5 (rs2115819) and LTA4H (rs2660845) genes were successfully typed. There was no difference between members of the general population (n = 200) and patients (n = 52) in each genotype frequency. Significant associations were found between SNP genotypes in the LTA4H gene and changes in PEF and FEV(1·0) . The PEF and FEV(1·0) responses to montelukast in the A/A genotypes (n = 4) for the LTA4H SNP were significantly higher than those in the G allele carriers (A/G+G/G) (n = 17). WHAT IS NEW AND CONCLUSION: Despite the small sample size, our results suggest that genetic variation in leukotriene pathway candidate genes contributes to variability in clinical responses to montelukast in Japanese patients with asthma.
Assuntos
Acetatos/farmacologia , Antiasmáticos/farmacologia , Araquidonato 5-Lipoxigenase/genética , Asma/tratamento farmacológico , Epóxido Hidrolases/genética , Quinolinas/farmacologia , Acetatos/uso terapêutico , Adulto , Idoso , Alelos , Antiasmáticos/uso terapêutico , Povo Asiático/genética , Asma/genética , Ciclopropanos , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Genótipo , Humanos , Japão , Antagonistas de Leucotrienos/farmacologia , Antagonistas de Leucotrienos/uso terapêutico , Leucotrienos/genética , Masculino , Pessoa de Meia-Idade , Pico do Fluxo Expiratório/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Quinolinas/uso terapêutico , Análise de Sequência de DNA , Sulfetos , Resultado do TratamentoRESUMO
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell-cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell- cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell- cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell- cell adhesion.
Assuntos
Adesão Celular , Proteínas de Ligação ao GTP/fisiologia , Transativadores , Actinas/fisiologia , Animais , Caderinas/metabolismo , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Tamanho Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Fosfoproteínas/metabolismo , Solubilidade , Transfecção , Proteína da Zônula de Oclusão-1 , beta Catenina , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteínas rho de Ligação ao GTPRESUMO
As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.
Assuntos
Divisão Celular , Dano ao DNA , Fase G2 , Proteínas Proto-Oncogênicas/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Linhagem Celular , Proliferação de Células , Fatores de Transcrição E2F/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Interferência de RNARESUMO
Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.
Assuntos
Antígenos de Superfície/metabolismo , Química Encefálica , Proteínas de Transporte/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/genética , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/química , Western Blotting , Células COS/fisiologia , Cálcio/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA Complementar , Exocitose/fisiologia , Isomerismo , Dados de Sequência Molecular , Proteínas Munc18 , Proteínas do Tecido Nervoso/química , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Células PC12 , Ligação Proteica/fisiologia , Proteínas R-SNARE , Ratos , Sintaxina 1RESUMO
Very few normal human peripheral blood T cells are capable of binding autologous erythrocytes to form rosettes, whereas in the T cell population activated by concanavalin A (Con A) the autorosette levels are markedly enhanced. Fractionation of the Con A-activated T cells with autologous erythrocytes into autorosetting and nonrosetting cells demonstrates that suppressor, but not helper, activity resides in the autorosetting population, whereas the reverse is true of the nonrosetting population. Both these activities are found to be Con A dependent. The Con A-induced human suppressor cells can be identified and separated from the Con A-induced human helper cells by the autorosette technique. Studies on the surface properties of autorosetting and nonrosetting T cells indicate that there is little correlation between the activated suppressor and helper T cell subsets defined by autorosette technique and either those defined by monoclonal antibodies (which are able to distinguish these subsets in the resting but not activated T cells) or those defined by Fc receptors. Since the autorosetting T cell population (which acts as suppressor cells) bears receptors for peanut agglutinin, the nature of Con A-induced human suppressor cells appears to be analogous to that of Con A-induced murine suppressor cells.
Assuntos
Formação de Roseta , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Antígenos de Superfície/análise , Autoantígenos/análise , Separação Celular/métodos , Concanavalina A/farmacologia , Eritrócitos/imunologia , Humanos , Cooperação LinfocíticaRESUMO
Patients with systemic lupus erythematosus (SLE) were found to have in their plasma antibodies specific for desialized T cells. Adsorption studies with intact or desialized T cells indicated that SLE anti-T cell antibodies consisted of two populations with different target cell specificities, one capable of recognizing unique determinants on desialized T cells and another able to bind to both intact and desialized T cells. Normal T cells did not remove the antibodies specific for desialized T cells. moreover, the antibodies to desialized T cells were not removed by adsorption with either desialized non-T cells or desialized erythrocytes. Thus, the antibodies to desialized T cells recognize a determinant that is unique to a T cell subset and also includes a sugar. Inhibition studies with various sugars indicated that lactose was the most potent inhibitor of antibody binding. The anti-desialized T cell antibody appears to recognize a T cell determinant which includes lactose, probably in the form of a beta-galactosyl residue, but which also includes additional T cell determinants. The antibodies to desialized T cells were found to bind preferentially to concanavalin A-induced autorosetting T cells, which had been already demonstrated to contain suppressor effector cells. Indeed, such antibodies were effective in eliminating suppressor effector function without interfering with T cells necessary for such activation (such as precursor or inducer cells). Finally, studies of patients with SLE yielded a highly significant correlation (r = 0.92) between impaired suppressor effector function of their cells and the presence of antibodies to desialized T cells in their plasma.
Assuntos
Especificidade de Anticorpos , Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T Reguladores/imunologia , Imunofluorescência , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Ácidos Siálicos/imunologia , Ácidos Siálicos/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The relationship between RNA synthesis and homologous pairing in vitro, catalyzed by RecA protein, was examined by using an established strand transfer assay system. When a short DNA duplex is mixed with single-stranded circles, RecA protein promotes the transfer of the minus strand of the duplex onto the complementary region of the plus-strand circle, with the displacement of the plus strand of the duplex. However, if minus-strand RNA is synthesized from the duplex pairing partner, joint molecules containing the RNA transcript, the plus strand of the DNA duplex, and the plus-strand circle are also observed to form. This reaction, which is dependent on RNA polymerase, sequence homology, and RecA protein, produces a joint molecule that can be dissolved by treatment with RNase H but not RNase A. Under these reaction conditions, product molecules form even when the length of shared homology between duplex and circle is reduced to 15 bp.
Assuntos
DNA Circular/química , DNA de Cadeia Simples/química , RNA/química , Recombinases Rec A/metabolismo , Recombinação Genética , Transcrição Gênica , Técnicas In Vitro , Homologia de Sequência do Ácido NucleicoRESUMO
RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome. Under these conditions there is no reaction leading to formation of joint molecule complexes. However, transcription on the chromatin template activates the formation of complexes. Reaction is dependent on RNA synthesis and DNA sequence homology and proceeds regardless of the direction of transcription.
Assuntos
Nucleossomos/fisiologia , Recombinases Rec A/metabolismo , Recombinação Genética , Transcrição Gênica , Sistema Livre de Células , Cromatina/ultraestrutura , RNA Polimerases Dirigidas por DNA/metabolismo , Técnicas In Vitro , Moldes GenéticosRESUMO
Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by various combinations of myoclonus epilepsy, ataxia, choreoathetosis and dementia. No specific therapy has been established and renal complication is rare. We report two cases of DRPLA with renal complications. Hematuria and proteinuria had gradually progressed for 2 and 13 years in these patients. Renal biopsy findings revealed focal glomerulosclerosis in one case and end-stage kidney disease in the other case. Angiotensin-converting enzyme inhibitor and angiotensin receptor II antagonist were administered to both patients, resulting in improved proteinuria and preserved renal function in one patient, while renal function continued to deteriorate in the other patient. Although renal complication is rare in patients with DRPLA, the presence of renal disease has to be suspected in patients with persistent proteinuria.
Assuntos
Glomerulosclerose Segmentar e Focal/complicações , Falência Renal Crônica/complicações , Epilepsias Mioclônicas Progressivas/complicações , Adulto , Feminino , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Falência Renal Crônica/patologia , MasculinoRESUMO
The antitumor drugs NB-506 and J-107088 are potent topoisomerase I inhibitors with an indolocarbazole structure. To clarify the factors involved in resistance to these drugs, we established two NB-506-resistant mouse fibroblast cell lines (LY/NR1 and LY/NR2), a human colon carcinoma cell line (HCT116/NR1), and a lung cancer cell line (PC13/NR1). These cell lines were highly resistant to NB-506 and J-107088, and LY/NR2 cells showed markedly reduced accumulation and strong efflux of NB-506, suggesting activation of a drug efflux pump in the resistant cells. To identify the molecules responsible for efflux of NB-506, we compared the gene expressions of the mouse resistant LY/NR1 cells, LY/NR2 cells, and their parental cells by oligonucleotide microarray. Of 34,020 genes analyzed, we found that an ATP-binding cassette transporter BCRP/MXR/ABCP (BCRP) gene showed the highest increase in the expression, 31-fold higher in the LY/NR2-resistant cells than in their parental cells. The selective overexpression of this gene was also detected in the two human resistant cell lines, suggesting the involvement of breast cancer resistant protein (BCRP) in the resistance and efflux of these drugs. Finally, a PC-13 cell line transfected with BCRP expression vector displayed 22- and 17-fold resistance to NB-506 and J-107088 and enhanced efflux activity of J-107088. However, the transfectants were not resistant to mitoxantrone or topotecan, the drugs previously thought to be the substrates of BCRP. Thus, our study presents a novel mechanism of drug resistance mediated by BCRP.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carbazóis/farmacocinética , Inibidores Enzimáticos/farmacocinética , Glucosídeos/farmacocinética , Indóis , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Transporte Biológico , Carbazóis/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , DNA Complementar/genética , DNA de Neoplasias/genética , Resistência a Múltiplos Medicamentos , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Glucosídeos/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Inibidores da Topoisomerase I , Transfecção , Células Tumorais CultivadasRESUMO
Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Amplificação de Genes , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib/farmacologia , Immunoblotting , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Compostos de Fenilureia/farmacologia , Piperazinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Rho small G protein regulates various actin-dependent cell functions. As to the functioning sites of Rho, Rho regulates formation of stress fibers and focal adhesions in many types of cultured cells, whereas we have shown that the association sites of actin filaments with the plasma membrane controlled by the ERM (Ezrin, Radixin, Moesin) family are the functioning sites of Rho in MDCK cells stably expressing myc-RhoA. We have investigated here the effect of microinjection of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, or guanosine 5'-(3-O-thio) triphosphate-bound active form of Rho on the intracellular localization of both the ERM family and vinculin, which is one of the structural proteins of focal adhesions, in wild type MDCK cells. The ERM family was preferentially localized at peripheral bundles of actin filaments which are localized at the outer edge of colonies of the cells, microvilli and low Ca2+-induced cortical bundles of actin filaments in wild type MDCK cells. Microinjection of Rho GDI or C3 inhibited the localization of the ERM family at both the peripheral bundles and the low Ca2+-induced cortical bundles. On the other hand, vinculin was localized at both focal adhesions and basal edges of the colonies of the cells, and microinjection of Rho GDI or C3 inhibited the localization of vinculin at both of these sites. These results indicate that activation of Rho is necessary for the association of both the ERM family and vinculin with the plasma membrane in wild type MDCK cells. Microinjection of the guanosine 5'-(3-O-thio) triphosphate-bound form of Rho induced an increase in the localization of vinculin at focal adhesions, but did not induce an increase in the localization of the ERM family at the plasma membrane, indicating that activation of Rho itself is sufficient only for the association of vinculin with the plasma membrane at focal adhesions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fatores de Transcrição/fisiologia , Vinculina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Adenosina Difosfato Ribose/metabolismo , Animais , Cálcio/metabolismo , Adesão Celular , Compartimento Celular , Linhagem Celular , Membrana Celular/metabolismo , Cães , Substâncias Macromoleculares , Microvilosidades/ultraestrutura , Proteínas rho de Ligação ao GTPRESUMO
Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Proteínas de Grupo de Alta Mobilidade/fisiologia , Histonas/fisiologia , Ustilago/enzimologia , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/metabolismo , CinéticaRESUMO
A gene encoding a Ustilago maydis Rad51 orthologue has been isolated, rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and gamma radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Ustilago/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Fúngicos , DNA Fúngico , Raios gama , Regulação da Expressão Gênica , Genes Fúngicos , Mitose , Dados de Sequência Molecular , Plasmídeos , Rad51 Recombinase , Recombinação Genética , Raios UltravioletaRESUMO
The isolation and characterization of homogeneous cell populations are of great importance for the analysis of gene expression, because normal tissues contain various types of cells, and the differences in the populations of isolated cells exert significant effects on gene expression analysis. Researchers have attempted to develop methods for the isolation of homogeneous cell populations, such as flow cytometry and mechanical dissection. However, the recent emergence of laser-assisted microdissection has revolutionized the isolation of single-cell populations from solid tissues. With the help of a cutting laser, laser microdissection can isolate tissues (cells) of interest without contamination from surrounding tissues with the microscopic visualization field. By combining laser microdissection and subsequent microarray technology, several studies have resulted in the identification of disease-related genes. In this review, we summarize the principle of laser microdissection and provide several successful examples of target-gene identification using the conventional method combining laser microdissection and microarray. Next, we discuss the practical drawbacks of the combinational method, such as the need for a large number of cells and the disturbance of the relative abundance of transcripts during RNA amplification. We introduce our modifications to combined laser microdissection and microarray for detection of disease-related genes; the technique is simple, yet practical and accurate. Finally, versatile applications of laser microdissection, not only to transcript expression analysis, but also to other genomics and proteomics analyses are, also presented.
Assuntos
Lasers , Microdissecção , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica , Animais , HumanosRESUMO
The use of defined or serum-free culture conditions during retroviral transduction of hematopoietic cells would be desirable for standardization and safety reasons, as well as potentially allowing greater expansion of progenitor cells. Retroviral vector supernatants were concentrated and purified via tangential flow filtration polyethylene glycol (PEG)-precipitation, and ultracentrifugation, allowing serum-free transductions at standard multiplicities of infection (moi). Protein content of transductions using these concentrated vectors was 5-6 logs lower than in standard transductions. Transduction efficiencies of these concentrated vector preparations added back to serum-free or serum-containing media were equivalent to standard retroviral supernatant transductions of CD34-enriched progenitors. Absolute progenitor (CFU-C) numbers at the end of transduction were higher in serum-free + concentrated virus transductions, as opposed to transductions in standard vector supernatants containing fetal calf serum.
Assuntos
Antígenos CD34/genética , Expressão Gênica , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/imunologia , Sequência de Bases , Meios de Cultura Livres de Soro , Primers do DNA , Células-Tronco Hematopoéticas/citologia , Humanos , Dados de Sequência Molecular , Retroviridae/genética , TransfecçãoRESUMO
To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.