RESUMO
We investigated the effects of two 5-HT(6) receptor antagonists on rat primary hepatocytes using a combined biochemical and toxicogenomics approach. Both compounds share the same pharmacological target, but displayed strikingly different toxicity profiles in pre-clinical animal studies: While R7199 caused hepatic steatosis in rats, no hepatotoxicity was observed with R0074. Here, we partially reproduced the steatosis findings seen in vivo using primary rat hepatocytes. Biochemical analyses and gene expression results generally supported the findings observed in the animal model and also allowed the differentiation of both compounds with regards to hepatotoxic potential. In particular, the induction of Cyp 2B and Cyp 3A1 directly correlates to the findings in the livers of treated animals. The effects on genes of the steroideogenic pathway relate to the deregulation of cholesterol homeostasis. We also observed the inhibition of beta-oxidation, indicating impaired lipid metabolism. Hence, gene expression analysis in combination with biochemical parameters can provide additional insight into the possible mechanisms underlying adverse events.
Assuntos
Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/toxicidade , Toxicogenética/métodos , Animais , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/metabolismo , Colesterol/metabolismo , Citocromo P-450 CYP2B1/efeitos dos fármacos , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A , Indução Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/patologia , Masculino , Oxirredução/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus. The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a Mr of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a Mr of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases. This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A. niger phytase.
Assuntos
6-Fitase/genética , Ascomicetos/genética , Genes Fúngicos/genética , 6-Fitase/química , Sequência de Aminoácidos , Ascomicetos/enzimologia , Aspergillus niger/genética , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Glicosilação , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
High-affinity receptors for human IFN-gamma were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high-affinity binding site for each cell. One cross-linked complex between IFN-gamma and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N-linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN-gamma and antibody epitopes characteristic of the extracellular domain of the IFN-gamma receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high-affinity IFN-gamma receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high-affinity IFN-gamma receptor protein. N-linked sugars may give structural stability to the IFN-gamma receptor and are unlikely to be directly involved in IFN-gamma binding.
Assuntos
Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Receptores Imunológicos/metabolismo , Anticorpos Monoclonais , Northern Blotting , Linhagem Celular , Humanos , Cinética , Leucemia , Linfoma , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Receptores Imunológicos/genética , Receptores Imunológicos/isolamento & purificação , Receptores de Interferon , Proteínas RecombinantesRESUMO
Serotonin is involved in disorders of the central nervous system; thus, specific 5-HT(6) receptor antagonists have therapeutic potential. Nevertheless, preclinical tests showed that Ro 65-7199 caused hepatic steatosis. Here, we investigated the hepatic effects of Ro 65-7199 and Ro 66-0074 using toxicogenomics. The profiles obtained after exposure of rats to both compounds clearly show that two pharmacologically closely related compounds with different toxicological profiles can be distinguished based on gene expression profiles. Moreover, side effects can be detected earlier with toxicogenomics than with conventional end points. A possible link between the sterol metabolic pathway, the induction of CYP2B, and the hepatic fat accumulation was also established. Summarizing, gene expression profiles allow both compounds to be distinguished according to their toxicity and provide mechanistic insights. The results clearly show the power of toxicogenomics as a tool for obtaining characteristic fingerprints at early time-points and for generating mechanistic hypotheses.
Assuntos
Perfilação da Expressão Gênica/métodos , Imidazóis/toxicidade , Indóis/toxicidade , Fígado/efeitos dos fármacos , Piridinas/toxicidade , Pirrolidinas/toxicidade , Receptores de Serotonina/fisiologia , Antagonistas da Serotonina/toxicidade , Compostos de Espiro/toxicidade , Sulfonamidas/toxicidade , Animais , Imidazóis/química , Imidazóis/farmacologia , Indóis/química , Indóis/farmacologia , Fígado/patologia , Masculino , Piridinas/química , Piridinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ratos , Ratos Wistar , Receptores de Serotonina/genética , Antagonistas da Serotonina/química , Antagonistas da Serotonina/farmacologia , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , SulfonasRESUMO
The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.
Assuntos
6-Fitase/genética , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Clonagem Molecular , 6-Fitase/isolamento & purificação , 6-Fitase/metabolismo , Sequência de Aminoácidos , Aspergillus niger/genética , Sequência de Bases , DNA Fúngico/genética , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transcrição Gênica , Transformação GenéticaRESUMO
The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.