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1.
J Cell Sci ; 123(Pt 20): 3558-65, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20930144

RESUMO

The activating transcription factor 3 (ATF3) gene is induced by a variety of signals, including many of those encountered by cancer cells. We present evidence that ATF3 is induced by TGFß in the MCF10CA1a breast cancer cells and plays an integral role for TGFß to upregulate its target genes snail, slug and twist, and to enhance cell motility. Furthermore, ATF3 upregulates the expression of the TGFb gene itself, forming a positive-feedback loop for TGFß signaling. Functionally, ectopic expression of ATF3 leads to morphological changes and alterations of markers consistent with epithelial-to-mesenchymal transition (EMT). It also leads to features associated with breast-cancer-initiating cells: increased CD24(low)-CD44(high) population of cells, mammosphere formation and tumorigenesis. Conversely, knockdown of ATF3 reduces EMT, CD24(low)-CD44(high) cells and mammosphere formation. Importantly, knocking down twist, a downstream target, reduces the ability of ATF3 to enhance mammosphere formation, indicating the functional significance of twist in ATF3 action. To our knowledge, this is the first report demonstrating the ability of ATF3 to enhance breast cancer-initiating cell features and to feedback on TGFß. Because ATF3 is an adaptive-response gene and is induced by various stromal signals, these findings have significant implications for how the tumor microenvironment might affect cancer development.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fator 3 Ativador da Transcrição/genética , Neoplasias da Mama/genética , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores de Hialuronatos/metabolismo , Immunoblotting , Imunoprecipitação , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Anal Biochem ; 390(1): 91-3, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19306836

RESUMO

Aberrant transglutaminase (TG) activity has been implicated in the pathology of numerous diseases, including Huntington's disease and Alzheimer's disease. To fully characterize the role of TGs in these disorders, it is important that simple quantifiable assays be made available. The most commonly used assay currently employed requires significant time and a radioactive substrate. The assay described here uses a biotinylated substrate in conjunction with a dot blot apparatus to eliminate the use of radioactive substrates and allows relative transglutaminase activity to be measured simultaneously with minimal sample preparation in a large number of samples containing purified enzyme, cell extracts, or tissue homogenates.


Assuntos
Medições Luminescentes/métodos , Transglutaminases/análise , Aminas/química , Animais , Biotina/análogos & derivados , Biotina/química , Biotinilação , Densitometria , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/metabolismo
3.
J Biomed Mater Res B Appl Biomater ; 103(4): 870-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25142105

RESUMO

Antibiotic-loaded bone cement is a primary option for treatment of orthopedic infections. Poly(methyl methacrylate) (PMMA) is a widely used cement that, when loaded with antibiotics in spacer or bead form, has been shown to reduce infection rates. However, PMMA is not resorbable and requires a second surgery for removal, while also acting as a potential foreign body for bacterial colonization. Alternatively, resorbable bone cements, such as calcium sulfate, have been proposed and present the advantage of being completely reabsorbed. It is unknown whether the antibiotic elution characteristics of absorbable bone cements are similar to PMMA. This study (1) characterized antibiotic elution from synthetic, highly purified calcium sulfate cement beads of varying sizes against pathogenic bacteria both in liquid culture and seeded on agar plates, (2) tested calcium sulfate beads against PMMA beads loaded with the same antibiotics, and (3) analyzed the structural differences between how PMMA and calcium sulfate bind to antibiotics. In every assay, the calcium sulfate beads performed as well as, or better than, the PMMA beads in inhibition of bacterial growth and elution of vancomycin in vitro with complete elution observed from calcium sulfate within three days. These data suggest that calcium sulfate, functions, as well as PMMA in the patient setting for infection control.


Assuntos
Antibacterianos , Sulfato de Cálcio , Portadores de Fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Polimetil Metacrilato , Staphylococcus epidermidis/crescimento & desenvolvimento , Infecção da Ferida Cirúrgica/tratamento farmacológico , Antibacterianos/química , Antibacterianos/farmacologia , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Sulfato de Cálcio/química , Sulfato de Cálcio/farmacologia , Polimetil Metacrilato/química , Polimetil Metacrilato/farmacologia
4.
J Biomed Mater Res B Appl Biomater ; 102(4): 667-73, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24155136

RESUMO

Treatments of osteomyelitis lag behind bacterial resistance to antibiotics. We tested different-sized calcium sulfate beads and their ability to elute multiple antibiotics in vitro as a possible method to improve the therapeutic delivery in patients. Two sizes of calcium sulfate beads (4.8 and 3.0 mm diameter) that contained vancomycin, tobramycin, or both were dissolved in phosphate-buffered saline, and the rate of dissolution by weight and antibiotic elution by the disc diffusion assay and high-pressure liquid chromatography were measured. The 4.8 mm beads showed significantly higher dissolution rates relative to the 3.0 mm beads (2.3 mg/day vs. 1.3 mg/day). While the vancomycin-loaded 4.8 mm beads eluted for a longer time relative to the 3.0 mm beads (20 days vs. 10 days), the smaller beads had threefold higher elution for the first 2 days, before dropping to near zero elution by day 4. The presence of tobramycin extended the elution of the vancomycin to day 40, which closely matches the recommended 6 weeks to treat orthopedic staphylococcus infections. These data suggest that size and content of the bead are variables that could affect their clinical success, and both could be exploited to tailor treatments of specific infections and injuries.


Assuntos
Antibacterianos/administração & dosagem , Sulfato de Cálcio/química , Tobramicina/administração & dosagem , Vancomicina/administração & dosagem , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Portadores de Fármacos , Composição de Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Microesferas , Tamanho da Partícula , Solubilidade , Tobramicina/farmacocinética , Tobramicina/farmacologia , Vancomicina/farmacocinética , Vancomicina/farmacologia
5.
Future Microbiol ; 9(8): 987-1007, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25302955

RESUMO

As the number of total joint arthroplasty and internal fixation procedures continues to rise, the threat of infection following surgery has significant clinical implications. These infections may have highly morbid consequences to patients, who often endure additional surgeries and lengthy exposures to systemic antibiotics, neither of which are guaranteed to resolve the infection. Of particular concern is the threat of bacterial biofilm development, since biofilm-mediated infections are difficult to diagnose and effective treatments are lacking. Developing therapeutic strategies have targeted mechanisms of biofilm formation and the means by which these bacteria communicate with each other to take on specialized roles such as persister cells within the biofilm. In addition, prevention of infection through novel coatings for prostheses and the local delivery of high concentrations of antibiotics by absorbable carriers has shown promise in laboratory and animal studies. Biofilm development, especially in an arthoplasty environment, and future diagnostic and treatment options are discussed.


Assuntos
Artroplastia de Substituição , Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas à Prótese/microbiologia , Animais , Antibacterianos/uso terapêutico , Aderência Bacteriana , Humanos , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/prevenção & controle
6.
J Clin Invest ; 123(7): 2893-906, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23921126

RESUMO

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.


Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Imunidade Adaptativa , Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Movimento Celular , Técnicas de Cocultura , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Macrófagos/imunologia , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise Multivariada , Transplante de Neoplasias , Células Neoplásicas Circulantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Transcriptoma , Carga Tumoral , Células Tumorais Cultivadas
7.
Antioxid Redox Signal ; 12(4): 435-43, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19659431

RESUMO

Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions, but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a "knock-in" mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1alpha mRNA is dramatically induced by mutant huntingtin, HIF-1alpha depletion by short interfering RNAs (siRNA) does not affect steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover, 3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIF-dependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity. Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1alpha gene expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing cell death in conditions such as Huntington's disease and Alzheimer's disease that are associated with metabolic stress in the central nervous system.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Doença de Huntington/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Animais , Morte Celular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/enzimologia , Nitrocompostos/toxicidade , Estresse Oxidativo , Pró-Colágeno-Prolina Dioxigenase/efeitos dos fármacos , Propionatos/toxicidade , RNA Mensageiro/metabolismo , Ratos
8.
EMBO Mol Med ; 2(9): 349-70, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20665636

RESUMO

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.


Assuntos
Proteínas de Ligação ao GTP/antagonistas & inibidores , Doença de Huntington/enzimologia , Doença de Huntington/genética , Transcrição Gênica , Transglutaminases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Citocromos c/genética , Citocromos c/metabolismo , Modelos Animais de Doenças , Drosophila , Metabolismo Energético , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Humanos , Doença de Huntington/metabolismo , Camundongos , Mitocôndrias/metabolismo , Nitrocompostos/toxicidade , Peptídeos/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Regiões Promotoras Genéticas , Propionatos/toxicidade , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo
9.
Biochemistry ; 44(21): 7830-43, 2005 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-15909997

RESUMO

Several active transglutaminase (TGase) isoforms are known to be present in human and rodent tissues, at least three of which, namely, TGase 1, TGase 2 (tissue transglutaminase), and TGase 3, are present in the brain. TGase activity is known to be present in the cytosolic, nuclear, and extracellular compartments of the brain. Here, we show that highly purified mouse brain nonsynaptosomal mitochondria and mouse liver mitochondria and mitoplast fractions derived from these preparations possess TGase activity. Western blotting and experiments with TGase 2 knock-out (KO) mice ruled out the possibility that most of the mitochondrial/mitoplast TGase activity is due to TGase 2, the TGase isoform responsible for the majority of the activity ([14C]putrescine-binding assay) in whole brain and liver homogenates. The identity of the mitochondrial/mitoplast TGase(s) is not yet known. Possibly, the activity may be due to one of the other TGase isoforms or perhaps to a protein that does not belong to the classical TGase family. This activity may play a role in regulation of mitochondrial function both in normal physiology and in disease. Its nature and regulation deserve further study.


Assuntos
Encéfalo/enzimologia , Mitocôndrias Hepáticas/enzimologia , Transglutaminases/isolamento & purificação , Transglutaminases/metabolismo , Animais , Western Blotting , Cadaverina/metabolismo , Cálcio/fisiologia , Radioisótopos de Carbono/metabolismo , Caseínas/metabolismo , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Fluoresceína/metabolismo , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Soros Imunes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Putrescina/metabolismo , Ensaio Radioligante , Partículas Submitocôndricas/enzimologia , Transglutaminases/deficiência , Transglutaminases/genética , Transglutaminases/imunologia
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