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1.
Mol Microbiol ; 73(4): 680-94, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19656291

RESUMO

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética
2.
EMBO Mol Med ; 12(6): e11164, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32302470

RESUMO

The angiopoietin (Ang)-Tie pathway has been intensely pursued as candidate second-generation anti-angiogenic target. While much of the translational work has focused on the ligand Ang2, the clinical efficacy of Ang2-targeting drugs is limited and failed to improve patient survival. In turn, the orphan receptor Tie1 remains therapeutically unexplored, although its endothelial-specific genetic deletion has previously been shown to result in a strong reduction in metastatic growth. Here, we report a novel Tie1 function-blocking antibody (AB-Tie1-39), which suppressed postnatal retinal angiogenesis. During primary tumor growth, neoadjuvant administration of AB-Tie1-39 strongly impeded systemic metastasis. Furthermore, the administration of AB-Tie1-39 in a perioperative therapeutic window led to a significant survival advantage as compared to control-IgG-treated mice. Additional in vivo experimental metastasis and in vitro transmigration assays concurrently revealed that AB-Tie1-39 treatment suppressed tumor cell extravasation at secondary sites. Taken together, the data phenocopy previous genetic work in endothelial Tie1 KO mice and thereby validate AB-Tie1-39 as a Tie1 function-blocking antibody. The study establishes Tie1 as a therapeutic target for metastasis in a perioperative or neoadjuvant setting.


Assuntos
Neoplasias , Receptor de TIE-1 , Angiopoietina-1 , Angiopoietina-2 , Animais , Deleção de Genes , Humanos , Camundongos , Neovascularização Patológica , Receptor de TIE-1/genética , Receptor TIE-2
3.
J Bacteriol ; 190(18): 6170-7, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18658270

RESUMO

Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrofenóis/metabolismo , Fenilacetatos/metabolismo , Fatores de Transcrição/metabolismo , Tiramina/análogos & derivados , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Oxirredução , Fenetilaminas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Fatores de Transcrição/genética , Tiramina/metabolismo
4.
J Clin Invest ; 113(11): 1571-81, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15173883

RESUMO

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2'-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic beta cell function.


Assuntos
Diabetes Mellitus/metabolismo , Fígado/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Peptídeos/metabolismo , Receptores de Glucagon/genética , Animais , Glicemia/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Camundongos , Oligodesoxirribonucleotídeos Antissenso/genética , Ratos
5.
Diabetes ; 54(6): 1846-53, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15919808

RESUMO

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.


Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Fígado/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Receptores de Glucocorticoides/metabolismo , Animais , Dexametasona/farmacologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperlipidemias/tratamento farmacológico , Linfopenia/induzido quimicamente , Linfopenia/fisiopatologia , Camundongos , Camundongos Obesos , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , Ratos
6.
DNA Seq ; 13(2): 77-83, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12180349

RESUMO

Nine thousand and eighty-eight base pairs of the chicken Hoxa 11 gene, including 8470 bases 5' of the translation start site were sequenced, and the characteristics of the upstream sequence investigated. Consistent with previous findings that middle repetitive elements are rare in the HoxA cluster, no repetitive elements were found other than simple oligonucleotide repeats. Multiple and pairwise alignments of the chicken upstream sequence with its human and mouse orthologs revealed multiple regions of 80% or higher homology across species. For the chicken, these regions were separated by sequences with no significant homology to human, mouse, or in most cases any other Genbank sequences. Selective clustering of transcription factor binding motifs was found to occur within the conserved homologous regions, suggesting evolutionary conservation of critical regulatory sequences. Of particular interest, seven conserved Cdx binding sites were found in the Hoxa 11 promoter, suggesting regulation by a non-clustered Caudal homeobox gene. Previous analysis of the mouse and human Hoxa 11 genes found a conserved antisense transcript, of unknown function. The chicken Hoxa 11 antisense strand included a conserved open reading frame capable of encoding 168 amino acids. Comparison of this region in mouse and chicken showed seven insertion/deletions, with each a multiple of three bases, thereby preserving open reading frame.


Assuntos
Elementos de DNA Transponíveis , DNA Antissenso/fisiologia , Proteínas de Homeodomínio/genética , Proteínas Oncogênicas/genética , Filogenia , Animais , Sequência de Bases , Sítios de Ligação , Galinhas , Sequência Conservada , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de DNA
7.
J Bacteriol ; 188(3): 874-81, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16428390

RESUMO

Microarray studies of the Escherichia coli response to nitric oxide and nitrosative stress have suggested that additional transcriptional regulators of this response remain to be characterized. We identify here the product of the yjeB gene as a negative regulator of the transcription of the ytfE, hmpA and ygbA genes, all of which are known to be upregulated by nitrosative stress. Transcriptional fusions to the promoters of these genes were expressed constitutively in a yjeB mutant, indicating that all three are targets for repression by YjeB. An inverted repeat sequence that overlaps the -10 element of all three promoters is proposed to be a binding site for the YjeB protein. A similar inverted repeat sequence was identified in the tehA promoter, which is also known to be sensitive to nitrosative stress. The ytfE, hmpA, ygbA, and tehA promoters all caused derepression of a ytfE-lacZ transcriptional fusion when present in the cell in multiple copies, presumably by a repressor titration effect, suggesting the presence of functional YjeB binding sites in these promoters. However, YjeB regulation of tehA was weak, as judged by the activity of a tehA-lacZ fusion, perhaps because YjeB repression of tehA is masked by other regulatory mechanisms. Promoters regulated by YjeB could be derepressed by iron limitation, which is consistent with an iron requirement for YjeB activity. The YjeB protein is a member of the Rrf2 family of transcriptional repressors and shares three conserved cysteine residues with its closest relatives. We propose a regulatory model in which the YjeB repressor is directly sensitive to nitrosative stress. On the basis of similarity to the nitrite-responsive repressor NsrR from Nitrosomonas europaea, we propose that the yjeB gene of E. coli be renamed nsrR.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Óxido Nítrico/farmacologia , Nitrito Redutases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Genes Reguladores/genética , Nitrito Redutases/genética
8.
Muscle Nerve ; 33(5): 609-18, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16421881

RESUMO

Transthyretin (TTR) amyloidosis, the most common form of hereditary systemic amyloidosis, is characterized clinically by adult-onset axonal neuropathy and restrictive cardiomyopathy. More than 85 mutations in transthyretin have been found to cause this hereditary disease. Since essentially all circulating TTR is of hepatic origin, orthotopic liver transplantation has been used as the only specific form of therapy. Unfortunately, in many patients amyloid deposition continues after orthotopic liver transplantation, indicating that mutant TTR is no longer required for progression of the disease after tissue deposits have been initiated. As a first step toward medical treatment of this disease, we have employed antisense oligonucleotides (ASOs) to inhibit hepatic expression of TTR. A transgenic mouse model carrying the human TTR Ile84Ser mutation was created and shown to express high levels of human mutant transthyretin. TTR ASOs suppressed hepatic TTR mRNA levels and serum TTR levels by as much as 80%. Suppression of hepatic synthesis of transthyretin may offer a medical treatment for transthyretin systemic amyloidosis.


Assuntos
Amiloidose Familiar/tratamento farmacológico , Amiloidose Familiar/genética , Inativação Gênica , Oligonucleotídeos Antissenso/uso terapêutico , Pré-Albumina/antagonistas & inibidores , Alanina Transaminase/sangue , Amiloidose Familiar/sangue , Animais , Aspartato Aminotransferases/sangue , Carcinoma Hepatocelular/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica/métodos , Isoleucina/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , Mutação/fisiologia , Pré-Albumina/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Serina/genética , Fatores de Tempo
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