RESUMEN

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

Asunto(s)

Inhibidores de 5-alfa-Reductasa , Azaesteroides/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Finasterida/farmacocinética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Azaesteroides/sangre , Azaesteroides/farmacología , Unión Competitiva , Células Cultivadas , Dutasterida , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/farmacología , Finasterida/sangre , Finasterida/farmacología , Insectos , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Testosterona/metabolismo , Factores de Tiempo , Transfección

RESUMEN

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

Asunto(s)

Baculoviridae/genética , ADN Recombinante/genética , Vectores Genéticos , Transducción Genética , Integración Viral , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Cricetinae , Genes Reporteros , Proteínas Fluorescentes Verdes , Hibridación Fluorescente in Situ , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN

RESUMEN

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Asunto(s)

Ácido Anhídrido Hidrolasas/química , ADN Helicasas/química , Replicación del ADN , Proteínas de Unión al ADN/química , Papillomaviridae/química , Proteínas Virales/química , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Antígenos Transformadores de Poliomavirus/metabolismo , Baculoviridae/genética , Células Cultivadas , Dicroismo Circular , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Insectos/citología , Insectos/virología , Ratones , Nucleósido-Trifosfatasa , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismo

RESUMEN

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Proteínas de Unión al ADN/inmunología , Vectores Genéticos , Nucleopoliedrovirus , Receptores Citoplasmáticos y Nucleares/inmunología , Factores de Transcripción/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Proteínas de Unión al ADN/genética , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Receptores X del Hígado , Ratones , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/inmunología , Nucleopoliedrovirus/patogenicidad , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción/genética , Proteínas Virales de Fusión/genética

RESUMEN

An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expression pattern of each of these receptors. We report the generation and characterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is ERbeta subtype-specific and capable of recognizing full-length human ERbeta as well as all of its known protein isoforms. ER15.64A, raised against a ERbeta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to the immunizing peptide and the full-length E. coli expressed ERbeta in ELISA and BIAcore assays. It also immunostained nuclei of Sf9 insect cells that were infected with an ERbeta-baculovirus. In Western analysis, ER15.64A recognized ERbeta1 and ERbeta2 proteins from a reticulocyte in vitro transcription/translation preparation. This antibody did not cross-react with recombinant ERalpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis. The specificity of ER15.64A should make this antibody a useful tool for monitoring expression of ERbeta and its isoforms at the protein level and should aid in distinguishing the pattern of ERbeta receptor expression from that of ERalpha.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Receptores de Estrógenos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Reacciones Cruzadas , Receptor beta de Estrógeno , Femenino , Humanos , Hibridomas , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Inmunohistoquímica , Ratones , Isoformas de Proteínas/inmunología , Receptores de Estrógenos/metabolismo , Transducción Genética , Células Tumorales Cultivadas

RESUMEN

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

Asunto(s)

Metaloendopeptidasas/genética , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Membrana Celular/enzimología , Citoplasma/enzimología , Humanos , Insectos , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Baculovirus expression vectors are widely used for expressing heterologous proteins in cultured insect cells. Recent advances include further development of the system for production of multi-subunit protein complexes, co-expression of protein-modifying enzymes to improve heterologous protein production, and additional applications of baculovirus display technology. The application of modified baculovirus vectors for gene expression in mammalian cells continues to expand.

Asunto(s)

Baculoviridae/genética , Insectos/virología , Mamíferos/virología , Proteínas Recombinantes/genética , Animales , Antígenos/genética , Antígenos/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/genética

RESUMEN

125I-monitor peptide binding was performed using frozen sections of the rat liver and gut and visualized using autoradiography. Saturable binding was observed in unidentified single cells in the liver and in the mucosa of the small intestine. Epidermal growth factor (EGF) and GTPgammaS did not inhibit 125I-monitor peptide binding indicating that the binding sites are not EGF receptors or G protein-coupled receptors. The liver binding site exhibited an affinity 3.7-4.4-fold higher than those in the small intestine. It has been established that intraluminal monitor peptide releases cholecystokinin from the small intestine. The present results indicate that monitor peptide may also have liver associated functions.

Asunto(s)

Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Intestino Delgado/química , Hígado/química , Receptores de Péptidos/aislamiento & purificación , Animales , Autorradiografía , Unión Competitiva , Colecistoquinina/metabolismo , Clonación Molecular , Factor de Crecimiento Epidérmico/metabolismo , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/metabolismo , Sustancias de Crecimiento/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Radioisótopos de Yodo , Hormonas Pancreáticas/genética , Hormonas Pancreáticas/metabolismo , Unión Proteica , Ratas , Inhibidor de Tripsina Pancreática de Kazal/genética , Inhibidor de Tripsina Pancreática de Kazal/metabolismo

RESUMEN

Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.

Asunto(s)

Baculoviridae/genética , Expresión Génica , Vectores Genéticos , Transformación Genética , Animales , Baculoviridae/ultraestructura , Células CHO , Células COS , Células Cultivadas , Cricetinae , Citomegalovirus/genética , Proteínas Fluorescentes Verdes , Células HeLa , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Roedores , Virus 40 de los Simios/genética

RESUMEN

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Asunto(s)

Expresión Génica , Proteínas Recombinantes/biosíntesis , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Clonación Molecular/métodos , Sistema Enzimático del Citocromo P-450/biosíntesis , Ligandos , Mamíferos , Melanóforos/citología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Saccharomyces cerevisiae , Transfección/métodos , Xenobióticos/metabolismo , Xenopus laevis

RESUMEN

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Asunto(s)

Baculoviridae/genética , Inmunoglobulina G/genética , Activadores Plasminogénicos/genética , Proteínas Recombinantes de Fusión/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Línea Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera

RESUMEN

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

Asunto(s)

Desintegrinas/genética , Metaloendopeptidasas/genética , Precursores de Proteínas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Clonación Molecular , Secuencia Conservada , Desintegrinas/aislamiento & purificación , Desintegrinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Porcinos

RESUMEN

We have developed a rapid in situ screening procedure that enables prescreening of hundreds of hybridomas in a 96-well format. The procedure involves fluorescence immunostaining of cells cultured in 96-well plates and the use of a fluorescence plate reader to detect reactive antibodies. Positive immunostaining in individual well, as denoted by elevated readings, is then confirmed by fluorescence microscopy. Using the method described here, we have successfully identified monoclonal antibodies that are specific to the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma). This assay is readily applicable for screening hybridomas raised against cell surface or intracellular antigens to aid in the initial identification of antibodies reactive in immunocytochemical procedures.

Asunto(s)

Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Secuencia de Aminoácidos , Animales , Células COS , Hibridomas/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/análisis , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Espectrometría de Fluorescencia , Factores de Transcripción/análisis , Factores de Transcripción/genética , Transfección

RESUMEN

We have discovered that 17beta-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5alpha-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5 alpha-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 degrees C, an initial finasteride-reductase complex is formed with a K(i)(app) of 11.9 +/- 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 +/- 0.01 s(-1). In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a K(i) of 6.8 +/- 0.2 nM. The reversible formation of a final complex, with an overall K(i) of 0.07 +/- 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 +/- 0.0001 s(-1) for the forward step and 0.00025 +/- 0.00006 s(-1) for the backward step. All rate constants for the two-step mechanism were obtained by using a general numerical integration method. The best fit values for the association and dissociation rate constants were 5.0 microM(-1) s(-1) and 0.033 +/- 0.008 s(-1), respectively, and the isomerization rate constants were 0.0035 +/- 0.007 s(-1) and 0.000076 +/- 0.000019 s(-1). These values correspond to an initial K(i) of 6.5 nM and an overall dissociation constant of 0.14 nM. The data presented here show that both finasteride and the 6-aza-steroid analogs are potent against type II 5alpha-reductase, although their mechanisms of inhibition are different.

Asunto(s)

Inhibidores de 5-alfa-Reductasa , Azaesteroides/química , Inhibidores Enzimáticos/química , Finasterida/química , Humanos , Isoenzimas/antagonistas & inhibidores , Cinética , Relación Estructura-Actividad

Asunto(s)

Sistema Enzimático del Citocromo P-450/genética , NADH NADPH Oxidorreductasas/genética , Animales , Baculoviridae/genética , Reactores Biológicos , Línea Celular , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Expresión Génica , Humanos , Insectos , Microsomas/enzimología , NADH NADPH Oxidorreductasas/biosíntesis , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADPH-Ferrihemoproteína Reductasa , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Spodoptera , Virología/métodos

RESUMEN

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.

Asunto(s)

Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Interleucina-1/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Bases , Línea Celular , Núcleo Celular/genética , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , AMP Cíclico/farmacología , ADN/metabolismo , Inducción Enzimática , Humanos , Interleucina-1/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia , Transducción de Señal

RESUMEN

IL-1 beta is a cytokine generally considered to be a major component involved in the pathogenesis of rheumatoid arthritis and other inflammatory diseases. Of the agents found in high concentrations in inflamed rheumatoid arthritis joints, TNF-alpha is among the most strongly implicated as an in vivo inducer of IL-1 beta. Here we report that in human PBMC and in a stable transfectant of the promonocytic cell line, THP-1, TNF-alpha indeed appears to be an inducer of IL-1 beta production, but only in the presence of dibutyryl cAMP or agents such as the PG that elevate intracellular cAMP levels. This TNF-alpha/cAMP pathway regulates IL-1 beta production at the level of transcription and requires a cAMP response element located between -2762 and -2755 bp in the upstream regulatory sequence of IL-1 beta. Because PG, which are known to elevate cAMP levels in vivo, and TNF-alpha are both found in significant quantities in the synovial fluid of rheumatoid arthritis joints, the observed synergistic up-regulation in IL-1 beta synthesis by TNF-alpha/cAMP (PG) may provide valuable insight into the potential pathways involved in the continuous production of IL-1 beta in the chronically inflamed joint.

Asunto(s)

AMP Cíclico/fisiología , Interleucina-1/biosíntesis , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/química , Secuencia de Bases , Bucladesina/farmacología , Línea Celular , Inducción Enzimática/efectos de los fármacos , Humanos , Isoenzimas/biosíntesis , Datos de Secuencia Molecular , Monocitos/metabolismo , Placenta/enzimología , Transcripción Genética/efectos de los fármacos

RESUMEN

Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.

Asunto(s)

Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Animales , Baculoviridae/genética , Catálisis , Línea Celular , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Activación Enzimática , Expresión Génica , Humanos , Técnicas In Vitro , Cinética , Microsomas/enzimología , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Mariposas Nocturnas , NADPH-Ferrihemoproteína Reductasa/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Troleandomicina/farmacología

Asunto(s)

Clonación Molecular/métodos , Técnicas de Cultivo/instrumentación , Glicoproteínas/biosíntesis , Nucleopoliedrovirus/genética , Proteínas Recombinantes de Fusión/biosíntesis , Cultivo de Virus/instrumentación , Animales , Western Blotting , División Celular , Línea Celular , Técnicas de Cultivo/métodos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicoproteínas/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Nucleopoliedrovirus/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera/citología , Inhibidores Tisulares de Metaloproteinasas , Cultivo de Virus/métodos

RESUMEN

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one (finasteride), which has been approved for treatment of benign prostatic hyperplasia, is shown here to be a slow time-dependent inhibitor of human steroid 5 alpha-reductase isozyme 1. This inhibition is characterized by an initial, fast step where the inhibitor binds to the enzyme followed by a slow step that leads to a final enzyme-inhibitor complex (EI*). No recovery of activity from this EI* complex was observed after dialysis for 3 days. The formation of EI* is diminished in the presence of a competitive, reversible inhibitor, indicating that the inhibition is active site-directed. At 37 degrees C and pH 7.0, the rate constant for the second, slow inhibition step, k3, is (1.40 +/- 0.04) x 10(-3) s-1 and the pseudo-bimolecular rate constant, k3/Ki, is (4.0 +/- 0.3) x 10(3) M-1 s-1. This latter rate constant is less than the value of 2.7 x 10(5) M-1 s-1 determined for the inhibition of 5 alpha-reductase 2 by finasteride [Faller, B., Farley, D., & Nick, H. (1993) Biochemistry 32, 5705-5710].(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Inhibidores de 5-alfa-Reductasa , Finasterida/farmacología , Sitios de Unión , Humanos , Cinética