RESUMEN
Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that inactivates a family of fatty acid amide molecules which are implicated in physiological processes such as pain and sleep. We cloned a 1.9 kb fragment of the 5'-untranslated region of the mouse FAAH gene into the pGL3 basic luciferase reporter vector and showed that this sequence has promoter activity in vitro. By primer extension analysis, we have determined the transcription start site to be 200 bases upstream of the ATG initiation codon and found that a TATA motif was absent. A number of putative response elements, including those for estrogen and glucocorticoids, were identified in this sequence. We have demonstrated that the estrogen and glucocorticoid receptors down-regulate transcriptional activity independent of their ligand. These data should help in understanding the mechanisms of FAAH gene transcription.
Asunto(s)
Amidohidrolasas/genética , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Células CHO , Células COS , Cricetinae , ADN/química , ADN/genética , Estrógenos/farmacología , Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Glucocorticoides/farmacología , Humanos , Luciferasas/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Sitio de Iniciación de la Transcripción , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.
Asunto(s)
Trastornos de Somnolencia Excesiva/inducido químicamente , Trastornos de Somnolencia Excesiva/fisiopatología , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiopatología , N-Glicosil Hidrolasas , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas de Plantas/administración & dosificación , Adenosina Desaminasa/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Línea Celular , Ritmo Circadiano/efectos de los fármacos , Cricetinae , Electroencefalografía , Citometría de Flujo , Hipotálamo/patología , Inmunotoxinas/administración & dosificación , Inmunotoxinas/química , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Microinyecciones , Narcolepsia/inducido químicamente , Narcolepsia/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/química , Receptores de Orexina , Orexinas , Proteínas de Plantas/química , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G , Receptores de Neuroquinina-1/biosíntesis , Receptores de Neuroquinina-1/genética , Receptores de Neuropéptido/biosíntesis , Receptores de Neuropéptido/genética , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Sueño/efectos de los fármacos , Toxinas Biológicas , Transfección , Grabación en VideoRESUMEN
Hypocretins 1 and 2 (also called orexins A and B, respectively) are hypothalamic neuropeptides that have recently been shown to be involved in the sleep disorder narcolepsy and possibly in the normal regulation of sleep and wake functions. These two peptides are derived from a single precursor molecule called prepro-hypocretin, also known as prepro-orexin. We have cloned a 450 bp fragment from the 5'-flanking region of the human prepro-hypocretin gene and demonstrated that this fragment has promoter activity in vitro. Deletions at the 5' end from -450 to -188 reduced the promoter activity by approximately 50%. Further deletion from the 5'-end to -69 almost completely abolished promoter activity. The 450 bp fragment contains a number of potential transcription factor binding sites, including an interferon (IFN) response element. Our studies demonstrate that alpha-IFN strongly inhibits the promoter activity of both 450 and 188 bp fragments in a dose-dependent manner. The inhibitory effect of alpha-IFN is consistent with recent studies which suggest that hypocretin 1/orexin A may be involved in modulating arousal states and with the literature indicating involvement of immune-related molecules in sleep regulation.
Asunto(s)
Interferón-alfa/metabolismo , Neuropéptidos/genética , Precursores de Proteínas/genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Genes Reporteros , Humanos , Interferón-alfa/farmacología , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Orexinas , Regiones Promotoras Genéticas , Precursores de Proteínas/metabolismo , Elementos de Respuesta , Eliminación de SecuenciaRESUMEN
Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación Enzimológica de la Expresión Génica , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Transactivadores/fisiología , Acetilcisteína/farmacología , Animales , Hidroxianisol Butilado/farmacología , Hipoxia de la Célula , Cloranfenicol O-Acetiltransferasa/antagonistas & inhibidores , Cloranfenicol O-Acetiltransferasa/genética , Depuradores de Radicales Libres/farmacología , Genes Reporteros , Humanos , Ratones , NAD(P)H Deshidrogenasa (Quinona)/genética , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
Squamous cell carcinoma of the oral cavity spreads by initial invasion of the laminin-rich basement membrane. We examined the adhesion and motility of human oral SCC cells and normal mucosal keratinocytes and found that the SCC cells readily attached and migrated on laminin 1 substrates but migrated poorly on collagen type I and fibronectin. The normal keratinocytes, however, adhered poorly to and were non-motile on laminin 1 yet readily and preferentially attached and migrated on fibronectin and collagen type I. Analysis with blocking anti-integrin antibodies showed that the SCC cells used the alpha 6 beta 1 complex to attach and migrate on laminin 1 and that this activity was confined to the E8 long arm fragment of laminin. Affinity chromatography on laminin-Sepharose columns revealed that the SCC cells, but not normal keratinocytes, expressed high levels of the alpha 6 beta 1 laminin 1 receptor. Metabolic pulse-chase analysis indicated that in contrast to the SCC cells, keratinocytes did not have a stable pool of beta 1 subunit precursor. Preferential pairing of alpha 6 with beta 4 and the deficiency in pre-beta 1 levels appear to account for the failure of keratinocytes to form significant alpha 6 beta 1 complex. Additionally, the presence of laminin 1 in co-coating experiments blocked keratinocyte adhesion to other immobilized ligands, such as collagen type I or fibronectin. This anti-adhesive effect seemed to reflect a general paralysis of cell adhesive function, since laminin 1 also diminished the adhesion of keratinocytes to substrates coated with immobilized anti-integrin subunit antibody. The inhibitory activity of laminin 1 resided in the E1' and E8 fragments, and not in the E3, E4 or G domains. Collectively, our results indicate that laminin 1 is a restrictive ligand for normal keratinocytes, apparently because of their failure to assemble and express the alpha 6 beta 1 complex or other functional laminin receptors and their sensitivity to the anti-adhesive activity of laminin itself. The elevated expression of alpha 6 beta 1 following malignant conversion of muscosal keratinocytes promotes their migration on laminin, a process important during invasion and metastasis.
Asunto(s)
Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/metabolismo , Encía/citología , Integrinas/metabolismo , Queratinocitos/citología , Laminina/metabolismo , Neoplasias de la Boca/patología , Unión Competitiva , Adhesión Celular , Movimiento Celular , Células Cultivadas , Fibronectinas/metabolismo , Expresión Génica , Humanos , Ligandos , ARN Mensajero/genéticaRESUMEN
In full-term newborns, permanent closure of the ductus arteriosus is associated with the formation of a neointima that is characterized by extracellular matrix deposition and smooth muscle cell migration. Transforming growth factor-beta (TGF-beta), a potent modulator of extracellular matrix deposition and smooth muscle cell migration, has been found to play a role in the remodeling associated with several forms of vascular disease. We examined the protein and mRNA expression of the three mammalian isoforms of TGF-beta (TGF-beta1, TGF-beta2, and TGF-beta3) during ductus arteriosus closure in full-term lambs. We found that the temporal changes and cellular localization of the proteins and mRNAs of all three TGF-beta isoforms were similar. TGF-beta proteins and mRNAs were present in very low levels in the late-gestation fetal ductus. Within 24 h of delivery, there was enhanced expression of TGF-beta in the newly forming neointima and outer muscle media; this continued to increase over the next 10 d. Increased expression of TGF-beta in the inner muscle media and adventitia lagged behind that of the neointima and outer muscle media. TGF-beta was not found in the luminal endothelial cells at any time. In contrast to the pattern described above, the appearance of TGF-beta protein differed from that of mRNA in the vasa vasorum of the ductus wall. After delivery, there was an increase in TGF-beta immunoreactivity in the smooth muscle cell layers of the vasa vasorum without any concurrent mRNA expression. The appearance of TGF-beta at the time of ductus closure suggests an important role for this growth factor in the reorganization of the ductus wall after birth.
Asunto(s)
Conducto Arterial/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Conducto Arterial/embriología , Conducto Arterial/ultraestructura , Expresión Génica , Técnicas para Inmunoenzimas , Hibridación in Situ , ARN Mensajero/metabolismo , Ovinos , Factor de Crecimiento Transformador beta/genéticaRESUMEN
We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.
Asunto(s)
Carcinoma/irrigación sanguínea , Neoplasias del Colon/irrigación sanguínea , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Neovascularización Patológica , Etanidazol/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Humanos , Hidrocarburos Fluorados , Hipoxia/metabolismo , Hibridación in Situ , Indicadores y Reactivos , Organoides , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Anatomical closure of the ductus arteriosus (DA) requires normally quiescent smooth muscle cells (SMC) to migrate out of the muscle media into the subendothelial space, forming intimal mounds that eventually coalesce to occlude the vessel's lumen. Transforming growth factor-beta 1 (TGF beta 1), a potent modulator of vascular SMC migration, is found in the wall of the closing DA. We examined the effect of TGF beta 1 on the migration of fetal lamb DA-SMC. Although TGF beta 1 has been shown to be a chemoattractant for other mesenchymal cells, it had no chemotactic effect on DA-SMC; furthermore, TGF beta 1 did not enhance the migration of DA-SMC (as has been reported for aortic SMC). Rather, incubating DA-SMC with TGF beta 1 for 22 h decreased the rate of migration of SMC on extracellular matrix substrata composed of fibronectin, vitronectin, laminin, and collagen I and IV. Exposure of DA-SMC to TGF beta 1 was associated with an increase in the formation of focal adhesion plaques (tight associations between the cells' surface and extracellular matrix). DA-SMC use integrin receptors to attach to and migrate on extracellular matrix components. The decrease in DA-SMC migration was not associated with a significant change in the profile of integrin receptors expressed by the cell. TGF beta 1 had little effect on overall DA-SMC integrin expression, except for a modest increase in the fibronectin receptor (alpha 5 beta 1 integrin). Rather, the decrease in migration and changes in cell morphology were associated with an increased ability of integrin receptors to associate with the cytoskeleton. TGF beta 1 appears to anchor the cell's cytoskeleton to the extracellular matrix, making the cells more adherent and less capable of migrating.
Asunto(s)
Conducto Arterial/citología , Músculo Liso Vascular/citología , Factor de Crecimiento Transformador beta/fisiología , Animales , Bovinos , Adhesión Celular/fisiología , Células Cultivadas , Quimiotaxis/fisiología , Conducto Arterial/embriología , Matriz Extracelular/fisiología , Humanos , Ratones , Músculo Liso Vascular/embriología , Conejos , Ratas , OvinosRESUMEN
The ability of verapamil to overcome resistance to adriamycin in a multidrug-resistant derivative of the V79 cell line (LZ), grown as multicellular spheroids or as monolayers, was examined. Verapamil was much less effective in overcoming resistance to adriamycin in spheroids than in monolayers. Verapamil increased the adriamycin content of cells grown as monolayers, but had no significant effect on the drug content of spheroids. This occurred in spite of the same mdr-I mRNA and protein levels in monolayers and spheroids. When the surviving fraction of cells was normalized to the cellular adriamycin content, cells both in monolayers and spheroids treated with verapamil were still more sensitive to adriamycin than their counterparts not treated with verapamil. The observed resistance of spheroids to adriamycin and verapamil sensitization may be caused by a drug-resistance mechanism that is functional only in spheroids, in addition to the activity of P-glycoprotein. Multicellular tissue architecture and cell-cell contact may play significant roles in this type of multidrug-resistance mechanism.
Asunto(s)
Resistencia a Múltiples Medicamentos , Células Tumorales Cultivadas/efectos de los fármacos , Verapamilo/farmacología , Animales , División Celular/efectos de los fármacos , Cricetinae , Cricetulus , Doxorrubicina/farmacocinética , Doxorrubicina/toxicidad , Ensayos de Selección de Medicamentos Antitumorales , Modelos BiológicosRESUMEN
Receptor binding studies have demonstrated the presence of an [3H]MK-801 ([3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate) binding site in human keratinocytes. The affinity found in keratinocytes was lower than that found in brain membranes. Northern blots identified mRNA in human keratinocytes and rat cardiocytes, as well as rat brain, that hybridized with high stringency to a probe for NMDAR1, an NMDA receptor subunit. In each tissue, mRNA that hybridized to another glutamate binding protein that might be part of an NMDA receptor complex, was also present. The presence of NMDA or NMDA-like receptors in keratinocytes and rat cardiocytes together with the low affinity [3H]MK-801 binding suggests that this protein may be a general channel forming protein that is present in many tissues, and forms specific receptors by interacting with additional subunits.
Asunto(s)
Queratinocitos/metabolismo , Miocardio/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Northern Blotting , Células Cultivadas , Maleato de Dizocilpina/farmacocinética , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/ultraestructura , Ligandos , Miocardio/citología , Miocardio/ultraestructura , N-Metilaspartato/farmacología , Fenciclidina/farmacología , ARN Mensajero/biosíntesis , Ratas , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
During permanent closure of the ductus arteriosus, smooth muscle cells migrate through the extracellular matrix (ECM) to form intimal mounds that occlude the vessel's lumen. Smooth muscle cells (SMC) migrate over surfaces coated with collagen in vitro. During the migration SMC also synthesize fibronectin (FN) and laminin (LN). Antibodies against FN and LN inhibit migration on collagen by 30% and 67%, respectively. Because of the apparent importance of LN in migration, we examined how SMC interact with LN and LN fragments (P1, E8, P1', E1', E3, E4, and G). Ductus SMC adhere to high concentrations of LN and two fragments of the molecule: P1 and E8. They use a unique set of integrin receptors to bind to LN (alpha 1 beta 1, alpha 6 beta 1 and alpha v beta 3), to P1 (alpha 1 beta 1, alpha v beta 3), and to E8 (alpha 6 beta 1, alpha v beta 3). The alpha v beta 3 integrin binds to the P1 fragment of LN in an RGD peptide-dependent manner, and to the E8 fragment in an RGD-independent manner; the RGD site on the P1 fragment probably is not available to the cell in intact LN. Antibodies against beta 1 integrins completely inhibit SMC adhesion to LN; antibodies against the alpha v beta 3 integrin do not block SMC adhesion to LN, but do prevent cell spreading. LN is also capable of interfering with SMC adhesion to other ECM components. The antiadhesive effect of LN is located in the E1' domain. Both exogenous and endogenous LN increase SMC motility on collagen I. The locomotion-promoting activity of LN resides in the E1' antiadhesive domain, and not in its adhesive (P1, E8) domains. LN causes a decrease in the number of focal contacts on collagen I. This might enable SMC to alter their mobility as they move through the extracellular matrix to occlude the ductus arteriosus lumen.
Asunto(s)
Colágeno , Conducto Arterial/citología , Laminina/fisiología , Músculo Liso Vascular/citología , Receptores de Laminina/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Movimiento Celular/efectos de los fármacos , Cromatografía de Afinidad , Matriz Extracelular/metabolismo , Fibronectinas/farmacología , Integrinas/inmunología , Integrinas/metabolismo , Laminina/farmacología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Conejos , Ratas , Ovinos/embriologíaRESUMEN
Integrin expression in squamous cell carcinomas have been reported to be down-regulated in vivo. Because integrins are molecules whose functions include the reorganization of cytoskeleton in response to the surrounding extracellular matrix, we have examined the expression of integrins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 microns and approximately 600 microns. Since larger A431 spheroids require epidermal growth factor for growth, we also investigated the effect of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using monoclonal antibodies specific for alpha 6, beta 1, and beta 4 subunits revealed a strong staining pattern in the periphery of the spheroids. The interior cells of the spheroids showed a moderate, positive reaction with the beta 1 antibody but significantly reduced from that at the periphery. Anti-alpha 2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha 6 and beta 1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha 6, beta 1, and beta 4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha 2, alpha 5, or beta 5 mRNA. Conversely, the expression of alpha v was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha 2, alpha 6, beta 1, and beta 4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of alpha v or beta 5. These results mimic the pattern of expression found in vivo and indicate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of integrin molecules.
Asunto(s)
Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/ultraestructura , Comunicación Celular/fisiología , Regulación hacia Abajo/fisiología , Integrinas/fisiología , Animales , Northern Blotting , Western Blotting , Carcinoma de Células Escamosas/secundario , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/fisiología , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Receptores de Laminina/fisiología , Células Tumorales CultivadasRESUMEN
Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) are known to alter the migratory and proliferative capacity of endothelial cells in vitro and to stimulate angiogenesis in vivo. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor expression and activity. We examined the ability of these growth factors to modulate the expression of specific integrins in human microvascular endothelial cells (MEC). Immunoprecipitation of metabolically labeled MEC showed that bFGF upregulated the biosynthesis of alpha 2, alpha 5, beta 1, and beta 3. bFGF induced an increase in the levels of mRNA for alpha 2 and beta 1. TGF-beta increased synthesis of alpha 2, alpha 5, and beta 1. These results suggest that bFGF and TGF-beta selectively alter integrin profiles and influence interactions of MEC with the extracellular matrix during neovascularization. In particular, the upregulation of the collagen/laminin receptor, alpha 2 beta 1, by bFGF may provide activated endothelial cells with an enhanced capacity to migrate through both their underlying basement membrane and the interstitial matrix.
Asunto(s)
Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Integrinas/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Integrinas/genética , ARN Mensajero/metabolismoRESUMEN
A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described. Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), beta-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon. Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression. High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light. The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins. CTAP-III remained stable for more than 6 h following decay of the inducing signal. The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli.
Asunto(s)
Factores de Coagulación Sanguínea/genética , Colicinas , Genes Sintéticos , Vectores Genéticos , Péptidos , Proteínas Recombinantes de Fusión/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Recombinante , Escherichia coli , Humanos , Datos de Secuencia Molecular , Operón , PlásmidosRESUMEN
A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.
Asunto(s)
Escherichia coli/genética , Compuestos Férricos/metabolismo , Bacterias Gramnegativas/genética , Hidroliasas/genética , Clonación Molecular , Cósmidos/genética , Gránulos Citoplasmáticos , Biblioteca de Genes , Prueba de Complementación Genética , Magnetismo , Mapeo RestrictivoRESUMEN
The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.
Asunto(s)
Bacterias/genética , Escherichia coli/genética , Rec A Recombinasas/genética , Bacterias/efectos de la radiación , Southern Blotting , Clonación Molecular , Cósmidos , Reparación del ADN , Escherichia coli/efectos de la radiación , Biblioteca de Genes , Genes Bacterianos , Prueba de Complementación Genética , Peso Molecular , Hibridación de Ácido Nucleico , Plásmidos , Mapeo Restrictivo , Rayos UltravioletaRESUMEN
Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro+ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.
Asunto(s)
Bacterias/genética , Escherichia coli/genética , Genes Bacterianos , Transcripción Genética , Bacterias/metabolismo , Clonación Molecular , Cósmidos , ADN Bacteriano , Prueba de Complementación Genética , Vectores Genéticos , Prolina/metabolismoRESUMEN
Sixty eight strains of Salmonella typhimurium were isolated during an outbreak of salmonellosis in the paediatric ward of Saadi hospital in Shiraz, Iran. Eleven of the strains were shown to produce a single type of colicin of group E. This colicin was identified as colicin E5. Most of the colicinogenic strains exhibited similar drug sensitivity and phage typing patterns and all were of the serotype S. typhimurium var. Copenhagen.
Asunto(s)
Colicinas/biosíntesis , Salmonella typhimurium/metabolismo , Antibacterianos/farmacología , Tipificación de Bacteriófagos , Niño , Preescolar , Infección Hospitalaria/microbiología , Humanos , Lactante , Recién Nacido , Irán , Plásmidos , Infecciones por Salmonella/microbiología , Salmonella typhimurium/clasificación , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genéticaRESUMEN
We analyzed the structural and functional relationships among independently cloned segments of the plasmid ColE1 region that regulates and codes for colicin E1 (cea), immunity (imm), and the mitomycin C-induced lethality function (lys). On the basis of physiological properties, restriction endonuclease mapping, and DNA sequence analysis, the following recombinant plasmids were determined to represent three major functional classes: pNP12 (cea+, imm+, lys+), pNP4 (cea+, imm+, lys-), and pNP6 (cea+, imm-, lys-). Our results have established the order, boundaries, and relative orientation of the three structural genes, the location of the promoter region for imm gene transcription, and the predicted amino acid sequences of the imm and lys gene products. Hydropathicity analysis suggests that both proteins have hydrophobic amino acid segments characteristic of membrane-associated proteins. A model for the structure and expression of the colicin E1 operon is proposed in which the cea and lys genes are expressed from a single inducible promoter that is controlled by the lexA repressor in response to the SOS system of Escherichia coli. The imm gene lies between the cea and lys genes and is expressed by transcription in the opposite direction from a promoter located within the lys gene. This arrangement of structural genes indicates that the transcriptional units for all three genes overlap. We suggest that the formation of anti-sense RNA may be an important element in the coordinate regulation of gene expression in this system.