RESUMO
Objective: To investigate the incidence and risk factors of systemic allergic reactions induced by subcutaneous immunotherapy (SCIT) in patients undergoing SCIT injections in Peking Union Medical College Hospital (PUMCH). Methods: This is a single center retrospective cohort study. Using the outpatient information system of PUMCH, the demographic information and injection-related reaction data of patients undergoing SCIT injection in Allergy Department of PUMCH from December 2018 to December 2022 were retrospectively analyzed to count the incidence and risk factors of systemic allergic reactions caused by SCIT. Mann-Whitney nonparametric test or chi-square test was used for single-factor analysis, and multiple logistic regression was used for multiple-factor analysis. Results: A total of 2 897 patients received 18 070 SCIT injections in Allergy Department during the four years, and 40 systemic allergic reactions occurred, with the overall incidence rate of 0.22%. The incidence of systemic allergic reaction was 0.37% when using imported dust mite preparation and 0.15% when using domestic multi-component allergen preparation. The risk factors significantly related with SCIT-induced systemic allergic reactions in patients using imported dust mite preparation were age less than 18 years old (OR=3.186,95%CI: 1.255-8.085), highest injection concentration (OR value could not be calculated because all patients with systemic reactions were injected with highest concentration), and large local reaction in previous injection (OR=22.264,95%CI: 8.205-60.411). The risk factors for SCIT-induced systemic allergic reactions in patients using domestic allergen preparation were 5 or more types of allergens (OR=3.455,95%CI: 1.147-10.402), highest injection concentration (OR=3.794,95%CI: 1.226-11.740) and large local reaction in previous injection (OR=63.577,95%CI: 22.248-181.683). However, SCIT injection in pollen allergic patients during the pollen season did not show a correlation with systemic allergic reaction. Conclusion: The incidence of SCIT-induced systemic allergic reactions was low in the Chinese patient population of this study. Patients with one or more risk factors, such as multiple allergen injection, highest injection concentration, large local reaction in previous injection, should be given high attention and vigilance against systemic allergic reactions.
Assuntos
Alérgenos , Dessensibilização Imunológica , Hipersensibilidade , Humanos , Povo Asiático , Dessensibilização Imunológica/efeitos adversos , Hipersensibilidade/epidemiologia , Estudos RetrospectivosRESUMO
Objective:The aim of this study is to evaluate the effectiveness and safety of China Savin pollen extract which was used for skin prick test (SPT) in the diagnosis of China Savin pollen allergy. Method:Patients with diagnosis of allergic diseases were collected from Allergy Department of Peking Union Medical College Hospital. All patients were given SPT with China Savin pollen extract, and the mean wheal diameter (MWD) was measured after 15 minutes. Receiver operating characteristic curve (ROC) analysis was performed based on the results of serum specific immunoglobulin E (sIgE). The effectiveness of SPT in the diagnosis of China Savin pollen allergy was evaluated under different diagnostic cutoff values. Adverse events were also recorded to evaluate the safety. Result:A total of 1 029 patients were enrolled in this study without drop out case. There were 1 007 patients in full analysis set (FAS) and 765 patients in per protocol analysis set (PPS). The elimination rate was 25.66%. The area under the ROC curve of FAS is 0.814 (95%CI: 0.788-0.839); which of PPS is 0.829 (95%CI: 0.801-0.857). Based on the ROC curve of PPS, the optimal and the 95% specificity diagnostic cutoff values of MWD were 3.25 mm and 4.75 mm respectively. Based on different diagnostic cutoff value (3.00, 3.25 and 4.75 mm), the sensitivities of SPT with China Savin pollen extract were 0.740 0 (95%CI: 0.701 6-0.778 4), 0.700 (95%CI: 0.659 8-0.740 2) and 0.532 (95%CI: 0.488 3-0.575 7) respectively, whereas the specificity was gradually increased in sequence, which was 0.769 8 (95%CI: 0.719 1-0.820 5), 0.826 4 (95%CI: 0.780 8-0.872 0) and 0.950 9 (95%CI: 0.924 9-0.976 9) respectively. There were 7 adverse events observed among 6 patients (rate: 0.583%, 6/1 029). The manifestation was mild. There was no severe adverse event. Conclusion:SPT with China Savin pollen extract is an effective and safe tool for the diagnosis of China Savin pollen allergy. The effectiveness of diagnosis could be improved based on integration of medical history and different diagnostic threshold values of SPT.
Assuntos
Alérgenos/efeitos adversos , Pólen/efeitos adversos , Rinite Alérgica Sazonal/diagnóstico , China , Humanos , Imunoglobulina E , Testes CutâneosRESUMO
BACKGROUND: Hereditary angioedema is a rare autosomal dominant disease, and its correlation between genotype and phenotype seems not to exist. So far, there are very few studies on Chinese population. We aimed to establish a Chinese genetic database of hereditary angioedema and investigated the potential correlation between genotype and phenotype. METHOD: All the eight exons and intron-exon boundaries of C1 inhibitor gene were detected in 48 unrelated families with HAE. The correlations between genotype and clinical parameters were evaluated by R statistical software. RESULTS: Thirty-five different mutations (25 of them were novel) and 7 SNPs (3 of them were novel) were identified. Significant difference was found in the level of C1 inhibitor antigen (P = 0.01793) between different groups of mutational types. The correlation between different groups of mutational types and the level of C1 inhibitor antigen (0.5047, P = 0.00027) was significant. The different groups of mutational types showed neither difference nor correlations of clinical parameters (severity score and the level of C1 inhibitor function). CONCLUSION: It appears that nonsense, frameshift, and mutations on Arg466 can cause lower level of C1 inhibitor antigen than missense and in-frame mutations; however, it does not affect severity of symptoms.
Assuntos
Angioedemas Hereditários/genética , Povo Asiático/genética , Proteína Inibidora do Complemento C1/genética , Mutação , Polimorfismo de Nucleotídeo Único , Genótipo , Humanos , FenótipoRESUMO
We report a 19-year-old man with thrombosis of the portal vein associated with a nephrotic syndrome. A computed tomography showed obstruction of the portal vein with prominent collaterals and cavernous transformation. This case is noteworthy as a report of nephrotic syndrome accompanied by extensive abdominal venous thrombosis and was cured successfully.
Assuntos
Síndrome Nefrótica/complicações , Veia Porta/patologia , Trombose Venosa/etiologia , Adulto , Anticoagulantes/uso terapêutico , Circulação Colateral , Humanos , Imunossupressores/uso terapêutico , Circulação Hepática , Masculino , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/tratamento farmacológico , Flebografia/métodos , Veia Porta/diagnóstico por imagem , Veia Porta/fisiopatologia , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Trombose Venosa/diagnóstico , Trombose Venosa/tratamento farmacológico , Trombose Venosa/fisiopatologiaRESUMO
Although Myc-interacting zinc-finger protein-1 (Miz-1) is known to be a poxvirus and zinc-finger (POZ) transcription factor required for Myc transcriptional repression, additional regulatory function of Miz-1 is less well understood. Using a yeast two-hybrid screen, we identified human alternate reading frame (ARF) protein as a novel interaction partner of Miz-1. The zinc-finger domain of Miz-1 is involved in its binding to ARF. In addition, we found that Miz-1 was able to interact with p53 through its DNA-binding domain, thus to diminish the binding of p53 to its target promoter and inhibit p53-mediated gene transcription. Interestingly, the Miz-1-regulated p53 transcriptional suppression does not require the presence of ARF or Mdm2. Importantly, ARF and p53 were found to competitively bind to Miz-1 in regulating p53-mediated transcription, and this conclusion was verified by both in vitro binding assay and competitive chromatin immunoprecipitation assay using a bona fide p53 endogenous Bax and Puma promoters. Thus, our study reveals that Miz-1 acts as a p53 suppressor by interfering with p53 DNA-binding ability, and ARF is able to counteract the suppression of Miz-1 on p53 by direct binding to Miz-1, suggesting that Miz-1 is a novel mediator in the ARF-p53 pathway.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação da Expressão Gênica , Humanos , Immunoblotting , Imunoprecipitação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
p120-ras GTPase-activating protein (rasGAP) associates with Ras and negatively regulates Ras signaling by stimulating the intrinsic rate of Ras GTPase activity. rasGAP also associates with other cellular signaling proteins which suggest that rasGAP may play a role in coordinating other signal transduction pathways. Disruption of rasGAP in vivo results in extensive apoptosis. Fas-mediated apoptosis results in the activation of caspases that cleave cellular substrates which are important for maintaining cytoplasmic and nuclear integrity. We show here that rasGAP is proteolytically cleaved by caspases early in Fas-induced apoptosis of Jurkat cells. rasGAP was also cleaved by DNA-damaging chemotherapeutic agents and TNF-related apoptosis inducing ligand (TRAIL), also known as Apo2L. Based on the size of the products generated by cleavage of deletion mutants of rasGAP we predict that cleavage of rasGAP occurs in the hydrophobic region and between the SH2(2) and ras-p21 interacting domain which would leave an intact ras-p21 interacting domain. Interestingly, cleavage of rasGAP in vitro enhanced rasGAP hydrolysis activity. Our results demonstrate that diverse apoptotic stimuli cause caspase-mediated cleavage of rasGAP early in apoptosis.
Assuntos
Apoptose , Caspases/metabolismo , Proteínas/metabolismo , Receptor fas/metabolismo , Sítios de Ligação , Caspase 3 , Caspase 7 , Proteínas Ativadoras de GTPase , Humanos , Hidrólise , Células Jurkat , Mapeamento de Peptídeos , Células Tumorais Cultivadas , Proteínas Ativadoras de ras GTPaseRESUMO
Lung epithelium plays a central role in modulation of the inflammatory response and in lung repair. Airway epithelial cells are targets in asthma, viral infection, acute lung injury, and fibrotic lung disease. Activated T lymphocytes release cytokines such as interferon-gamma (IFN-gamma) that can cooperate with apoptotic signaling pathways such as the Fas-APO-1 pathway to induce apoptosis of damaged epithelial cells. We report that IFN-gamma alone and in combination with activation of the Fas pathway induced apoptosis in A549 lung epithelial cells. Interestingly, the corticosteroid dexamethasone was the most potent inhibitor of IFN-gamma- and IFN-gamma plus anti-Fas-induced apoptosis. IFN-gamma induced expression of an effector of apoptosis, the cysteine protease interleukin-1 beta-converting enzyme, in A549 cells. Dexamethasone, in contrast, induced expression of an inhibitor of apoptosis, human inhibitor of apoptosis (hIAP-1), also known as cIAP2. We suggest that the inhibition of epithelial cell apoptosis by corticosteroids may be one mechanism by which they suppress the inflammatory response.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Interferon gama/farmacologia , Pulmão/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Receptor fas/fisiologia , Anticorpos/farmacologia , Caspase 1 , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/biossíntese , DNA/biossíntese , Células Epiteliais/efeitos dos fármacos , Proteína Ligante Fas , Humanos , Pulmão/citologia , Pulmão/fisiologia , Glicoproteínas de Membrana/biossíntese , Timidina/metabolismo , Receptor fas/biossíntese , Receptor fas/imunologiaRESUMO
Apoptotic cells undergo characteristic morphological changes that include detachment of cell attachment from the substratum and loss of cell-cell interactions. Attachment of cells to the extracellular matrix and to other cells is mediated by integrins. The interactions of integrins with the extracellular matrix activates focal adhesion kinase (FAK) and suppresses apoptosis in diverse cell types. Members of the tumor necrosis family such as Fas and Apo-2L, also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in both suspension and adherent cells through the activation of caspases. These caspases, when activated, cleave substrates that are important for the maintenance of nuclear and membrane integrity. In this study, we show that FAK is sequentially cleaved into two different fragments early in Apo-2L-induced apoptosis. We also demonstrate that FAK cleavage is mediated by caspases and that FAK shows unique sensitivity to different caspases. Our results suggest that disruption of FAK may contribute to the morphological changes observed in apoptotic suspension and adherent cells.
Assuntos
Apoptose , Caspases , Moléculas de Adesão Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Reguladoras de Apoptose , Caspase 1 , Caspase 3 , Caspase 6 , Caspase 7 , Moléculas de Adesão Celular/química , Inibidores de Cisteína Proteinase/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/química , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismoRESUMO
Airway epithelial cells modulate the inflammatory response in asthmatic, allergic and fibrotic lung diseases through the secretion of cytokines that regulate the movement and activation of inflammatory cells. Mast cells play an important role in the pathogenesis of these lung diseases. In this study we report that normal airway epithelial cells express stem cell factor which is a critical mediator of mast cell growth and differentiation and that transforming growth factor-beta inhibits secretion of stem cell factor by airway epithelial cells.
Assuntos
Brônquios/metabolismo , Fator de Células-Tronco/biossíntese , Brônquios/citologia , Brônquios/efeitos dos fármacos , Diferenciação Celular , Divisão Celular , Extratos Celulares , Linhagem Celular , Linhagem Celular Transformada , Membrana Celular , Citosol , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Expressão Gênica , Humanos , Mastócitos/citologia , RNA Mensageiro/análise , Fator de Células-Tronco/química , Fator de Células-Tronco/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin. At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr. The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships. Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain. Here, we present a mutational analysis of this domain using n-chimaerin sequences. Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified. Seven of the mutants, including deletions, still possessed GAP activity for Rac1. Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1. IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants. Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Família Multigênica , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Quinases , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Quimerina 1 , DNA , Escherichia coli , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcr , Alinhamento de Sequência , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTPRESUMO
We have analyzed the function of the CYP1A1 promoter using in vitro transcription. Using nuclear extracts from HeLa cells, we found that transcription from the promoter in vitro was constitutive. Transcription in vitro was not increased by prior exposure of the HeLa cells to the inducer 2,3,7, 8-tetrachlorodibenzo-p-dioxin nor by the inclusion of a dioxin-responsive enhancer in the DNA template. Analyses of mutants revealed that a TATAAA sequence and two recognition motifs for CCAAT box transcription factor/nuclear factor I contributed to the constitutive activity of the promoter in vitro.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Bases , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Células HeLa/efeitos dos fármacos , Células HeLa/fisiologia , Humanos , Dibenzodioxinas Policloradas/farmacologia , TATA Box , Moldes GenéticosRESUMO
We have reviewed the transcriptional regulation of the CYP1A1 gene, which encodes the cytochrome P450IA1 enzyme. TCDD induces CYP1A1 transcription via a receptor- and enhancer-dependent mechanism that involves multiple protein-DNA interactions. The system provides an example of enzyme regulation at the level of gene transcription.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Receptores de Droga/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Linhagem Celular , Cromatina/efeitos dos fármacos , Cromatina/fisiologia , Elementos Facilitadores Genéticos/efeitos dos fármacos , Indução Enzimática , Neoplasias Hepáticas Experimentais , Camundongos , Receptores de Hidrocarboneto Arílico , Receptores de Droga/efeitos dos fármacosRESUMO
We have developed a homologous in vitro transcription system that requires (i) 2,3,7,8-tetrachlorodibenzo-p-dioxin (called TCDD or dioxin), (ii) the Ah receptor, and (iii) a dioxin-responsive enhancer for activity. Unfractionated nuclear extracts from mouse hepatoma cells contain an inhibitor and fail to direct transcription in vitro. However, following phosphocellulose chromatography and reconstitution, the fractionated nuclear extract directs accurate transcription in vitro, using as a template the promoter/enhancer region from the mouse cytochrome P1-450 gene (Cyp1a1) linked to a "G-free cassette" (which generates a transcript with no guanosine residues). Extracts from TCDD-treated cells exhibit higher activity than extracts from untreated cells when transcribing a template containing both the promoter and enhancer but not when transcribing a template containing the promoter alone. Extracts from Ah receptor-defective cells fail to direct in vitro transcription in a TCDD-inducible fashion. A regulatory element that contains two binding sites for the liganded Ah receptor plus a truncated Cyp1a1 promoter suffices to direct TCDD-inducible, Ah receptor-dependent transcription in vitro. The inducible, receptor-dependent, enhancer-dependent properties of this system make it appropriate for analyzing in vitro the mechanism of dioxin action and the function of the Ah receptor.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Dibenzodioxinas Policloradas/farmacologia , Receptores de Droga/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Neoplasias Hepáticas Experimentais , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico , Moldes Genéticos , Transcrição Gênica/efeitos dos fármacosRESUMO
In a previous publication (Wen, L.-P., and Fulco, A. J. (1987) J. Biol. Chem. 262, 6676-6682), we described the cloning of the gene encoding cytochrome P-450BM-3, a catalytically self-sufficient fatty acid monooxygenase induced by barbiturates in Bacillus megaterium. We have now subcloned a 1.6-kilobase segment of DNA from this cloned gene that includes the barbiturate-responsive regulatory region as well as 88 bases encoding the NH2-terminal portion of cytochrome P-450BM-3. From this, we generated two series of 5' and 3' deletion derivatives and examined their effects on gene expression. When the 1.6-kilobase fragment or the 1.3-kilobase 5'----3' deletion derivative is inserted into Escherichia coli on a vector containing a promoterless chloramphenicol acetyltransferase (CAT) gene with the sequence encoding the NH2-terminal portion of P-450BM-3 placed immediately in front of the CAT gene, CAT activity is constitutive and unaffected by pentobarbital. On the other hand, the basal level of CAT is low in B. megaterium transformed by the same construct but is strongly inducible by pentobarbital. Furthermore, the multicopy plasmid containing this regulatory region causes a dramatic decrease in both the basal and pentobarbital-induced expression of chromosomally encoded P-450BM-3 in B. megaterium. This competition effect, unlike CAT expression, is independent of the orientation of the regulatory DNA segment in the plasmid. Removal of 0.3 kilobase or more from the 3' end of the 1.6-kilobase segment of DNA or 0.6 kilobase from the 5' end abolishes the competition effect and also eliminates basal and inducible CAT expression in B. megaterium. In transformed E. coli, constitutive CAT expression is maintained when as little as 0.3 kilobase of DNA from the 3' end of the 1.6-kilobase segment is inserted in the correct orientation in front of the CAT gene. The data are consistent with the hypothesis that the synthesis of cytochrome P-450BM-3 in B. megaterium is under positive control and requires gene interaction with at least one trans-acting factor, presumably a protein, to activate transcription from the P-450BM-3 promoter. The binding of this putative protein is mediated by at least two regulatory regions (R1 and R2) that span about 1 kilobase of the 5'-flanking region of the gene.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias , Barbitúricos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Oxigenases de Função Mista/genética , Bacillus megaterium/genética , Ligação Competitiva , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Desoxirribonuclease HindIII , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Oxigenases de Função Mista/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Pentobarbital/farmacologia , Plasmídeos , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transformação BacterianaRESUMO
Cytochrome P-450BM-3 (P-450BM-3) from Bacillus megaterium incorporates both a P-450 and an NADPH:P-450 reductase in proteolytically separable domains of a single, 119-kDa polypeptide and functions as a fatty acid monooxygenase independently of any other protein. A 5-kilobase DNA fragment which contains the gene encoding P-450BM-3 was sequenced. A single continuous open reading frame starting at nucleotide 1541 of the 5-kilobase fragment correctly predicted the previously determined NH2-terminal protein sequences of the trypsin-generated P-450 and reductase domains and, in toto, predicted a mature polypeptide of 1,048-amino acid residues with Mr = 117,641. The trypsin site was found at arginine residue 471. The P-450 domain is most similar (about 25%) to the fatty acid omega-hydroxylases of P-450 family IV, while the reductase domain exhibits some 33% sequence similarity with the NADPH:P-450 reductases of mammalian liver. Both the P-450 and reductase domains of P-450BM-3 define new gene families but contain highly conserved segments which display as much as 50% sequence similarity with P-450s and reductases of eukaryotic origin. The mRNA for P-450BM-3 was found by S1 mapping to be 3,339 +/- 10 nucleotides in length. In the accompanying paper, two regions in the 1.5 kilobases 5' to the P-450BM-3 coding region have been implicated in the regulation of P-450BM-3 gene expression.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias , Sistema Enzimático do Citocromo P-450/genética , DNA Bacteriano/genética , Oxigenases de Função Mista/genética , Sequência de Aminoácidos , Bacillus megaterium/genética , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Códon , Sistema Enzimático do Citocromo P-450/metabolismo , Enzimas de Restrição do DNA , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Peso Molecular , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Fragmentos de Peptídeos , RNA Mensageiro/genética , Transcrição Gênica , TripsinaRESUMO
In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160-7169, 1986; Ibid., 262: 6683-6690, 1987) we described the characterization of a catalytically self-sufficient 119,000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this polypeptide (cytochrome P-450BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676-6682, 1987). We have now compared authentic P-450BM-3 from B. megaterium and putative P-450BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.
Assuntos
Bacillus megaterium/genética , Proteínas de Bactérias/análise , Sistema Enzimático do Citocromo P-450/genética , Genes Bacterianos , Plasmídeos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Recombinação Genética , TripsinaRESUMO
In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.
Assuntos
Bacillus megaterium/genética , Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Bacterianos , Genes , Oxigenases/genética , Fenobarbital/farmacologia , Bacillus megaterium/efeitos dos fármacos , Bacillus megaterium/enzimologia , Sistema Enzimático do Citocromo P-450 , Indução Enzimática , Vetores Genéticos , Oxigenases/biossíntese , PlasmídeosRESUMO
In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843-850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.