RESUMO
With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.
Assuntos
Anti-Inflamatórios/química , Lactonas/química , Animais , Anti-Inflamatórios/farmacocinética , Desenho de Fármacos , Humanos , Interleucina-6/metabolismo , Lactonas/síntese química , Lactonas/farmacocinética , Camundongos , Microssomos Hepáticos/metabolismoRESUMO
Expression of the insulin gene is highly specific to pancreatic beta cells and is upregulated mainly by PDX-1 and BETA2/NeuroD depending on the extracellular glucose concentration. However, its downregulation has not been well studied. Reporter gene analyses using pancreatic HIT-T15 cells revealed that the glucose-dependent insulin promoter activity was blocked by glucocorticoids, dexamethasone (DEX) and hydrocortisone, in a dose-dependent manner. After the addition of DEX (20 nM) to HIT-T15 cells, a decrease of insulin mRNA was observed at 12-24 h, followed by a decline of insulin protein at 48 h. Expressions of PDX-1 and BETA2/NeuroD decreased within 2 h. HES-1, a potent negative regulator of bHLH-type transcription factors, was found to be expressed in HIT-T15 cells, and its expression was increased 6 h after the addition of DEX. Overexpression of HES-1 suppressed the insulin promoter activity in a dose-dependent manner. These results suggest that glucocorticoids impair insulin synthesis in HIT-T15 cells by decreasing PDX-1 and BETA2/NeuroD and that enhancement of HES-1 expression is involved in this regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucocorticoides/farmacologia , Proteínas de Homeodomínio/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Transativadores/metabolismo , Animais , Células Cultivadas , Cricetinae , Proteínas de Ligação a DNA/genética , Dexametasona/farmacologia , Proteínas de Homeodomínio/genética , Hidrocortisona/farmacologia , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Transativadores/genéticaRESUMO
Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway, which plays an important role in glucose toxicity and cellular insulin resistance. Thus, the mechanisms by which GFAT expression is regulated under physiological and pathological conditions are of interest in connection with diabetes. In this study, we cloned the 5'-flanking region of the mouse GFAT2 gene and characterized its promoter activity. Sequence analysis revealed several putative regulatory elements Sp1, a CCAAT box, AP-1 and AP-2, but no TATA box. Transfection experiments showed that the 5'-flanking region between -2462 to +38 relative to the transcription start site of the GFAT2 gene drives transcription in NIH3T3 cells and that the fragment from -141 to -9 has the highest transcription activity. Reporter assays using deletion and mutant variants suggested that the Sp1 sites at positions -83 to -78 and -22 to -17 both play an important role in the basal promoter activity of the mouse GFAT2 gene. Electrophoretic mobility shift assay showed DNA-protein binding at both Sp1 sites. We also compared the promoter activities of mouse GFAT1 and GFAT2 in several cell lines.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Regiões Promotoras Genéticas/genética , Células 3T3 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Clonagem Molecular , DNA/química , DNA/genética , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Íntrons , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição Sp1/metabolismo , Células Tumorais CultivadasRESUMO
Aggrecanase-1, also known as ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), cleaves at the Glu373-Ala374 site of aggrecan, thereby indicating aggrecan degradation. It is thought that ADAMTS4 plays a pivotal role in inflammatory joint diseases and cartilage degradation. To elucidate the mechanisms of regulation of ADAMTS4 gene expression, we cloned the 5'-flanking region of the human ADAMTS4 gene and characterized its promoter activity by means of reporter assay using porcine chondrocytes and NIH3T3 cells. Reporter gene analysis using deletion variants suggested that the region between -383 and +10 relative to the tentative transcription start site is necessary for full promoter activity; this region contains one Sp1 and three AP2 sites. In addition, the segment between -726 and -384 appears to contain silencer element(s). A complete deletion mutant of the nuclear factor I (NFI) binding site at -441 to -429 resulted in recovery of the promoter activity in chondrocytes, but not in NIH3T3 cells. Thus, the NFI site is involved in negative regulation of the human ADAMTS4 promoter activity in chondrocytes.
Assuntos
Regiões 5' não Traduzidas/genética , Proteínas de Ligação a DNA , Metaloendopeptidases/genética , Fatores de Transcrição , Células 3T3 , Proteínas ADAM , Proteína ADAMTS4 , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/genética , Condrócitos/metabolismo , Regulação da Expressão Gênica/genética , Genes Reporter , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFI , Proteínas Nucleares , Pró-Colágeno N-Endopeptidase , Regiões Promotoras Genéticas/genética , Deleção de Sequência , Suínos , Proteína 1 de Ligação a Y-BoxRESUMO
A cellular disintegrin, also called MDC and ADAM is a recently discovered gene family that encodes protein with disintegrin-like and metalloprotease-like domains. We have reported the identification of human cDNAs encoding novel ADAM family proteins that we named MDC2 and MDC3 because of their structural similarity to the MDC (Sagane, K. et al., 1998. Biochem. J. 334, 93-98). The Human Gene Nomenclature Committee assigned the gene symbols ADAM11 for the MDC, ADAM22 for the MDC2 and ADAM23 for the MDC3. Here we report the isolation of three novel murine cDNAs encoding the proteins closely related to the human ADAM11, ADAM22 and ADAM23. Their chromosomal locations in the mouse were identified by interspecies backcross mapping. The loci of these murine ADAM genes were in good accordance with the location of each human ortholog, ADAM11, ADAM22 and ADAM23. These findings suggest that three murine cDNAs that we have isolated are the murine ADAM11, ADAM22 and ADAM23 cDNAs. Northern blot analysis shows that all of these three murine ADAMs were highly expressed in the mouse brain. The structures of these ADAM proteins strongly suggest that they could function as integrin receptors. The implications of the cellular disintegrins in neural development are discussed.
Assuntos
Desintegrinas , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Proteínas do Tecido Nervoso/genética , Proteínas ADAM , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , DNA Complementar/análise , Integrinas/genética , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces pleiotropic inflammatory reactions. Soluble TNFalpha is released from its membrane-bound precursor by TNFalpha converting enzyme (TACE)/a disintegrin and metalloproteinase 17 (ADAM17). We have recently cloned the mouse TACE complete cDNA and the 5' flanking promoter region of the gene. Two versions of the mouse TACE cDNA having a coding region of 2541bp were obtained: one was about 4.1kb and the other was 4.4kb in length, in which only the length of the 3' UTR was different. Rapid amplification of 5' cDNA ends suggested that there were multiple transcriptional start sites. From the coding sequence, 827 amino acids were deduced which were 91.9 % identical to those of human TACE. Northern blot analysis indicated that the major transcript was approx. 4.4kb product in the mouse. The mouse TACE mRNA was ubiquitously expressed, and was particularly high in the lung. The proximal promoter contained multiple AP2 and Sp1 transcription factor binding sites and included a GC box and a CCAAT box, but lacked a consensus TATA box. Reporter gene analysis using RAW264.7 cells showed that the fragment at nt -290 to -1 from the translation start site has a strong promoter activity, and appeared to be essential for transcription of the mouse TACE mRNA. Finally, we found that the mouse TACE promoter at nt -2304 to -1 also worked in NIH3T3, 3T3L1 and C6 cell lines.
Assuntos
Metaloendopeptidases/genética , Regiões Promotoras Genéticas , Regiões 3' não Traduzidas , Células 3T3 , Proteínas ADAM , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Transcrição Gênica , Células Tumorais CultivadasAssuntos
Mapeamento Cromossômico/métodos , Cromossomos/genética , Metaloendopeptidases/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animais , Cromossomos Humanos Par 2/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICRAssuntos
Cromossomos Humanos Par 15/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Proteínas ADAM , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 15/ultraestrutura , Cricetinae , Primers do DNA/genética , Humanos , Células Híbridas , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Esclerose Múltipla/etiologia , Esclerose Múltipla/genética , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.