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1.
mBio ; 10(3)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239382

RESUMO

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Proteínas de Ligação a DNA/metabolismo , Lipoproteínas/metabolismo , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Southwestern Blotting , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Matriz Extracelular de Substâncias Poliméricas/genética , Lipoproteínas/genética , Espectrometria de Massas , Staphylococcus aureus/fisiologia , Eletricidade Estática
2.
Nucleic Acids Res ; 46(20): 10649-10668, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30445637

RESUMO

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3'OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3'OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.


Assuntos
DNA Fúngico/genética , DNA de Cadeia Simples/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Southwestern Blotting/métodos , Mapeamento Cromossômico/métodos , Quebras de DNA de Cadeia Simples , Clivagem do DNA , DNA Fúngico/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequências de Repetição em Tandem , Transcrição Gênica
3.
Methods Mol Biol ; 1334: 85-99, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26404144

RESUMO

Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-binding protein can be identified.


Assuntos
Southwestern Blotting/métodos , Proteínas de Ligação a DNA/química , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética/métodos , DNA/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Fatores de Transcrição/química , Fatores de Transcrição/genética
4.
Methods Mol Biol ; 1314: 63-71, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26139255

RESUMO

Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).


Assuntos
Northern Blotting/métodos , Southwestern Blotting/métodos , Western Blotting/métodos , DNA/metabolismo , Metiltransferases/metabolismo , RNA/metabolismo , Animais , Sondas de DNA/metabolismo , Humanos , Ligação Proteica
5.
Cancer Prev Res (Phila) ; 8(6): 552-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25813524

RESUMO

Non-small cell lung cancer (NSCLC) represents almost 85% of total diagnosed lung cancer. Studies have shown that combination of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors is effective against various cancers, including lung cancer. However, optimizing the synergistic dose regime is very difficult and involves adverse side effects. Therefore, in this study, we have shown that cucurbitacin B (CuB), a single bioactive triterpenoid compound, inhibits both DNMTs and HDACs starting at a very low dose of 60 nmol/L in NSCLC H1299 cells. The CuB-mediated inhibition of DNMTs and HDACs in H1299 cells leads to the reactivation of key tumor suppressor genes (TSG) such as CDKN1A and CDKN2A, as well as downregulation of oncogenes c-MYC and K-RAS and key tumor promoter gene (TPG), human telomerase reverse transcriptase (hTERT). The upregulation of TSGs and downregulation of TPG were consistently correlated with the alterations in their promoter methylation and histone modifications. This altered expression of TPG and TSGs is, at least in part, responsible for the inhibition of cellular proliferation and induction of cellular apoptosis in NSCLC. Furthermore, CuB treatment significantly inhibited the tumor incidence and multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, which was associated with the induction of apoptosis and inhibition of hyperproliferation in the lung tissues. Together, our study provides new insight into the CuB-mediated epigenetic alterations and its chemotherapeutic effects on lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/prevenção & controle , Nitrosaminas/toxicidade , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Southwestern Blotting , Western Blotting , Carcinógenos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Imunoprecipitação da Cromatina , Metilação de DNA/efeitos dos fármacos , Feminino , Genes Neoplásicos , Histona Desacetilases/química , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos A , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
6.
Nat Genet ; 46(10): 1072-80, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25151354

RESUMO

The ability to examine all chromatids from a single meiosis in yeast tetrads has been indispensable for defining the mechanisms of homologous recombination initiated by DNA double-strand breaks (DSBs). Using a broadly applicable strategy for the analysis of chromatids from a single meiosis at two recombination hotspots in mouse oocytes and spermatocytes, we demonstrate here the unidirectional transfer of information-gene conversion-in both crossovers and noncrossovers. Whereas gene conversion in crossovers is associated with reciprocal exchange, the unbroken chromatid is not altered in noncrossover gene conversion events, providing strong evidence that noncrossovers arise from a distinct pathway. Gene conversion frequently spares the binding site of the hotspot-specifying protein PRDM9, with the result that erosion of the hotspot is slowed. Thus, mouse tetrad analysis demonstrates how unique aspects of mammalian recombination mechanisms shape hotspot evolutionary dynamics.


Assuntos
Evolução Molecular , Meiose/genética , Oócitos/metabolismo , Recombinação Genética/genética , Espermatócitos/metabolismo , Animais , Southwestern Blotting , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Troca Genética , Feminino , Conversão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Modelos Genéticos , Oócitos/citologia , Espermatócitos/citologia
7.
Biochem Mol Biol Educ ; 42(5): 443-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25099485

RESUMO

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats.


Assuntos
Southwestern Blotting/métodos , Biologia Molecular/métodos , Resolução de Problemas , Inquéritos e Questionários , Southern Blotting/métodos , Western Blotting/métodos , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Imunoprecipitação/métodos , Biologia Molecular/educação , Aprendizagem Baseada em Problemas/métodos , RNA Polimerase II/metabolismo , Terminologia como Assunto
8.
Proc Natl Acad Sci U S A ; 110(48): 19408-13, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24218554

RESUMO

Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.


Assuntos
Adaptação Biológica/fisiologia , DNA Helicases/metabolismo , DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Sequência de Bases , Southwestern Blotting , Western Blotting , DNA Helicases/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Mitocondrial/fisiologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/farmacologia , Dados de Sequência Molecular , Miócitos Cardíacos/fisiologia , Superóxido Dismutase/genética
9.
PLoS One ; 8(9): e76397, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086737

RESUMO

The major obstacle of successful tumor treatment with carboplatin (CBP) is the development of drug resistance. In the present study, we found that following treatment with CBP the amount of platinum which enters the human laryngeal carcinoma (HEp2)-derived CBP-resistant (7T) cells is reduced relative to the parental HEp2. As a consequence, the formation of reactive oxidative species (ROS) is reduced, the induction of endoplasmic reticulum (ER) stress is diminished, the amount of inter- and intrastrand cross-links is lower, and the induction of apoptosis is depressed. In HEp2 cells, ROS scavenger tempol, inhibitor of ER stress salubrinal, as well as gene silencing of ER stress marker CCAAT/enhancer-binding protein (CHOP) increases their survival and renders them as resistant to CBP as 7T cell subline but did not influence the survival of 7T cells. Our results suggest that in HEp2 cells CBP-induced ROS is a stimulus for ER stress. To the contrary, despite the ability of CBP to induce formation of ROS and activate ER stress in 7T cells, the cell death mechanism in 7T cells is independent of ROS induction and activation of ER stress. The novel signaling pathway of CBP-driven toxicity that was found in the HEp2 cell line, i.e. increased ROS formation and induction of ER stress, may be predictive for therapeutic response of epithelial cancer cells to CBP-based therapy.


Assuntos
Carboplatina/uso terapêutico , Carcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Neoplasias Laríngeas/tratamento farmacológico , Transdução de Sinais/fisiologia , Apoptose/efeitos dos fármacos , Southwestern Blotting , Western Blotting , Carcinoma/fisiopatologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cinamatos , Óxidos N-Cíclicos , Primers do DNA/genética , Inativação Gênica , Humanos , Neoplasias Laríngeas/fisiopatologia , Platina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Marcadores de Spin , Tioureia/análogos & derivados
10.
Biochim Biophys Acta ; 1832(12): 2232-43, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23999007

RESUMO

Non-proliferative proteinuric diseases are the most common primary glomerular disorders causing end-stage renal disease. These disorders may associate low level glomerular inflammation and podocyte expression of inflammatory mediators. However, the factors regulating podocyte expression of inflammatory mediators in vivo in non-immune disorders are poorly understood. We have now explored the regulation and role of TWEAK receptor Fn14 in mediating glomerular inflammation in cultured podocytes and in experimental and human non-immune proteinuria. Transcriptomics disclosed Fn14 and MCP-1 mRNA upregulation in glomeruli from patients with focal segmental glomerulosclerosis, as well as a correlation between the expression of both transcripts. Increased glomerular Fn14 and MCP-1 mRNA was confirmed in a second focal segmental glomerulosclerosis cohort and was also observed in membranous nephropathy. In human non-proliferative proteinuric kidney diseases podocytes displayed Fn14 and MCP-1 expression and NFκB activation. Podocyte Fn14 was increased in murine protein overload-induced proteinuria. In Fn14 knock-out mice with protein overload-induced proteinuria, glomerular and periglomerular macrophage infiltrates were reduced, as were MCP-1 mRNA and podocyte MCP-1 staining and podocyte numbers preserved as compared to wild-type counterparts. Adenovirus-mediated overexpression of TWEAK increased periglomerular macrophage infiltration in mice without prior kidney injury. In cultured podocytes inflammatory cytokines increased Fn14 mRNA and protein levels. TWEAK activated NFκB and increased MCP-1 mRNA and protein, an effect prevented by the NFκB inhibitor parthenolide. In conclusion, Fn14 activation results in NFκB-mediated pro-inflammatory effects on podocytes that may be relevant for the pathogenesis of non-proliferative proteinuric kidney disease of non-immune origin.


Assuntos
Inflamação/metabolismo , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Southwestern Blotting , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Citocina TWEAK , Citocinas/genética , Citocinas/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Inflamação/genética , Inflamação/patologia , Nefropatias/genética , Nefropatias/patologia , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Podócitos/patologia , Proteinúria/genética , Proteinúria/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor de TWEAK , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismo
11.
PLoS One ; 7(7): e40617, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22808207

RESUMO

In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.


Assuntos
Diferenciação Celular , Regiões de Interação com a Matriz , Proteínas Associadas à Matriz Nuclear/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Especificidade de Anticorpos/imunologia , Sequência de Bases , Southwestern Blotting , Western Blotting , Linhagem Celular Tumoral , DNA de Neoplasias/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Lamina Tipo B/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo
12.
Toxicology ; 300(3): 121-31, 2012 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-22706169

RESUMO

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. Our present study shows that, for human keratinocytes (HaCaT) cells, a low concentration of arsenite activates extracellular signal-regulated kinases (ERKs), which leads to up-regulation of nuclear factor κB (NF-κB) binding to DNA and to elevated, NF-κB-dependent expression of mot-2 (a p53 inhibitor) and survivin (an inhibitor of apoptosis). Activation of p53 is blocked, and neoplastic transformation is enhanced. Inhibition of ERKs reduces cell proliferation and neoplastic transformation. In contrast, a high concentration of arsenite activates c-Jun N-terminal kinases (JNKs), positive regulators of p53, by binding to p53 and preventing its murine double minute 2 (mdm2)-mediated degradation. The elevated levels of p53 lead to repair of DNA damage and apoptosis. Inhibition of JNKs increases DNA damage but decreases apoptosis. By identifying a mechanism whereby ERKs and JNKs-mediated regulation of the p53-survivin signal pathway is involved in the biphasic effects of arsenite on human keratinocytes, our data expand understanding of arsenite-induced cell proliferation, neoplastic transformation, DNA damage, and apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Arsenitos/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Dano ao DNA , Proteínas Inibidoras de Apoptose/biossíntese , Queratinócitos/efeitos dos fármacos , Compostos de Sódio/toxicidade , Proteína Supressora de Tumor p53/biossíntese , Animais , Southwestern Blotting , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/biossíntese , Humanos , Imunoprecipitação , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Proteínas Mitocondriais/biossíntese , Neoplasias/induzido quimicamente , Neoplasias/metabolismo , Neoplasias/patologia , Reação em Cadeia da Polimerase em Tempo Real , Survivina
13.
Anal Biochem ; 427(2): 133-8, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22633908

RESUMO

Synthesis of (GT)5-tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our previous publication, we showed that the purification of the c-jun promoter using lambda exonuclease digestion of polymerase chain reaction (PCR) produced DNA with single-stranded tails. Asymmetric PCR can also produce tailed single strands that can be annealed to yield the desired promoter. An effective method uses asymmetric PCR and double digestion. After PCR, first a restriction enzyme, in this case SacII, cuts duplex strands remaining after asymmetric PCR, leaving 5' phosphoryl ends susceptible to a second digestion with lambda exonuclease to effectively degrade any duplex. The resulting single strands are then annealed to produce a duplex DNA with a single-stranded (GT)5 tail at the 3' end of each strand of the duplex. Unlike the previously described method, this novel procedure produces the desired tailed promoter devoid of any untailed duplex.


Assuntos
Cromatografia de Afinidade/métodos , DNA/análise , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Transcrição Gênica , Southwestern Blotting , DNA/genética , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Eletroforese em Gel Bidimensional , Exonucleases/genética , Exonucleases/metabolismo , Genes jun/genética , Humanos , Vírus da Leucemia Murina de Moloney/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
14.
Methods Mol Biol ; 786: 315-34, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21938635

RESUMO

Transcription factors regulate transcription by binding to regulatory regions of genes including the promoter. Few of the transcription factors are well characterized, and few promoters have been described in detail. New methods have been developed to improve both transcription factor and promoter characterization, some of which are discussed here. Trapping methodology applicable to both individual transcription factors and intact transcription complexes are described, as well as 2D gel electrophoresis, Southwestern blotting, and basic liquid chromatography/tandem mass spectrometry methodology. These methods have proved useful in the study of transcriptional regulation.


Assuntos
Proteômica/métodos , Fatores de Transcrição/metabolismo , Southwestern Blotting , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Espectrometria de Massas , Regiões Promotoras Genéticas/genética
15.
PLoS One ; 6(10): e27123, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22073128

RESUMO

BACKGROUND: Transforming growth factor type-ß (TGF-ß)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-ß signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-ß signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: TGF-ß1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-ß1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-ß1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-ß1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs. CONCLUSIONS: PARP1 is indispensable for TGF-ß1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-ß/Smad3 pathway.


Assuntos
Músculo Liso Vascular/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Benzamidas/farmacologia , Southwestern Blotting , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Fenantrenos/farmacologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/genética , Transativadores , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/genética
16.
J Chromatogr A ; 1218(39): 7003-15, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-21880322

RESUMO

Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3×10(7) HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes by proteomic methods.


Assuntos
Southwestern Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Proteômica/métodos , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas Estimuladoras de Ligação a CCAAT/química , Núcleo Celular/química , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray/métodos , Fator de Transcrição AP-1/química , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo
17.
Genesis ; 49(11): 835-40, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21630416

RESUMO

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild-type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild-type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations.


Assuntos
Animais Geneticamente Modificados/genética , Clonagem de Organismos/métodos , Cães/genética , Técnicas de Transferência de Genes , Proteínas Luminescentes/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Southwestern Blotting , Cães/sangue , Cães/metabolismo , Feminino , Fluorescência , Inseminação Artificial/métodos , Proteínas Luminescentes/genética , Masculino , Reação em Cadeia da Polimerase , Gravidez , Progesterona/sangue , Reprodução , Transgenes , Proteína Vermelha Fluorescente
18.
Ir J Med Sci ; 180(3): 709-14, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20535600

RESUMO

PURPOSE: To investigate whether topical application of hesperin affords protection to Balb/C mice epidermis from UVB-induced cyclobutane pyrimidine dimers (CPDs). METHODS: A DNA damage model of UVB irradiation-induced mice epidermis was established. The immunohistochemical staining and southwestern dot blotting were used for CPDs detection; western blotting was used for P53 detection. RESULTS: Topical application of hesperidin on Balb/C mice skin significantly decreased the amount of epidermal CPDs 24 and 48 h after 180 mJ/cm(2) of UVB irradiation as compared to untreated mice. UVB-induced p53 expression was more pronounced in hesperidin-treated mice epidermis compared to that of untreated mice. CONCLUSION: Taken together, these results suggest that topical hesperidin application promotes DNA photo-damage repair. Hesperidin is therefore a promising protective substance against UVB radiation.


Assuntos
Dano ao DNA/efeitos dos fármacos , Hesperidina/uso terapêutico , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta/efeitos adversos , Administração Tópica , Animais , Southwestern Blotting , Western Blotting , Enzimas Reparadoras do DNA/fisiologia , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Hesperidina/administração & dosagem , Hesperidina/química , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/fisiologia , Proteína Supressora de Tumor p53/metabolismo
19.
J Chromatogr A ; 1217(45): 7177-81, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20926088

RESUMO

Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized.


Assuntos
Fosfatase Alcalina/metabolismo , Southwestern Blotting/métodos , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Isótopos de Fósforo/metabolismo , Animais , Bovinos , Núcleo Celular/química , DNA/química , Proteínas de Ligação a DNA/química , Reutilização de Equipamento , Células HEK293 , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Isótopos de Fósforo/química , Fosforilação , Polivinil , Dodecilsulfato de Sódio , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
20.
BMC Genomics ; 11: 386, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20565887

RESUMO

BACKGROUND: Chromatin in the nucleus of all eukaryotes is organized into a system of loops and domains. These loops remain fastened at their bases to the fundamental framework of the nucleus, the matrix or the scaffold. The DNA sequences which anchor the bases of the chromatin loops to the matrix are known as Scaffold/Matrix Attachment Regions or S/MARs. Though S/MARs have been studied in yeast and higher eukaryotes and they have been found to be associated with gene organization and regulation of gene expression, they have not been reported in protists like Giardia. Several tools have been discovered and formulated to predict S/MARs from a genome of a higher eukaryote which take into account a number of features. However, the lack of a definitive consensus sequence in S/MARs and the randomness of the protozoan genome in general, make it a challenge to predict and identify such sequences from protists. RESULTS: Here, we have analysed the Giardia genome for the probable S/MARs predicted by the available computational tools; and then shown these sequences to be physically associated with the nuclear matrix. Our study also reflects that while no single computational tool is competent to predict such complex elements from protist genomes, a combination of tools followed by experimental verification is the only way to confirm the presence of these elements from these organisms. CONCLUSION: This is the first report of S/MAR elements from the protozoan parasite Giardia lamblia. This initial work is expected to lay a framework for future studies relating to genome organization as well as gene regulatory elements in this parasite.


Assuntos
DNA de Protozoário/genética , Trato Gastrointestinal/parasitologia , Genômica , Giardia lamblia/genética , Regiões de Interação com a Matriz/genética , Animais , Southwestern Blotting , Bovinos , Genoma de Protozoário/genética , Laboratórios , Espectrometria de Massas , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Reação em Cadeia da Polimerase
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