RESUMEN
OBJECTIVE: Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. METHOD: The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. RESULTS: Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRalpha and LXRbeta, and their heterodimeric partners retinoid X receptor (RXR)alpha and RXRbeta were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. CONCLUSIONS: Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.
Asunto(s)
Cartílago Articular/metabolismo , Proteínas de Unión al ADN/metabolismo , Osteoartritis/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Adulto , Anciano , Citocinas/farmacología , ADN Complementario/metabolismo , Proteínas de Unión al ADN/agonistas , Humanos , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado , Persona de Mediana Edad , Receptores Nucleares Huérfanos , Proteoglicanos/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores X Retinoide/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.
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Proteínas/genética , Proteínas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Encéfalo/embriología , Mapeo Cromosómico , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Ojo/embriología , Hibridación in Situ , Sistema Nervioso/embriología , Notocorda/metabolismo , Señales de Clasificación de Proteína , Cola (estructura animal)/embriologíaRESUMEN
We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.
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Proteínas Aviares , Cisteína/química , Mesodermo/metabolismo , Páncreas/embriología , Biosíntesis de Proteínas , Proteínas/química , Glándula Tiroides/embriología , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Clonación Molecular , ADN Complementario/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Hibridación in Situ , Pulmón/embriología , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularAsunto(s)
Fusión Artificial Génica/métodos , Proteínas Recombinantes/genética , Tiorredoxinas/genética , Animales , Clonación Molecular/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli , Vectores Genéticos , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/aislamiento & purificación , Humanos , Ratones , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Tiorredoxinas/biosíntesisAsunto(s)
Clonación Molecular/métodos , ADN Complementario/aislamiento & purificación , Glicósido Hidrolasas/genética , Señales de Clasificación de Proteína/genética , Secuencia de Bases , Cartilla de ADN , ADN Complementario/genética , Electroporación , Escherichia coli/genética , Biblioteca de Genes , Vectores Genéticos , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/biosíntesis , Saccharomyces cerevisiae/genética , beta-FructofuranosidasaRESUMEN
TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.
Asunto(s)
Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Proteínas de Dominio T Box , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Endodermo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Sustancias de Crecimiento/química , Sustancias de Crecimiento/metabolismo , Mesodermo/metabolismo , Microcefalia/genética , Datos de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/químicaRESUMEN
H174 is a new member of the CXC-chemokine family. A cDNA probe containing the entire H174 coding region recognized a predominant inducible transcript of approximately 1.5 kb expressed in interferon (IFN) activated astrocytoma and monocytic cell lines. H174 message can be induced following IFN-alpha, IFN-beta, or IFN-gamma stimulation. H174 message was also detected in IFN treated cultures of primary human astrocytes, but was absent in unstimulated astrocytes. H174, like IP10 and Mig, lacks the ELR sequence associated with the neutrophil specificity characteristic of most CXC-chemokines. Preliminary experiments suggest H174, IP10 and Mig are independently regulated. Recombinant H174 is a weak chemoattractant for monocyte-like cells. H174 can also stimulate calcium flux responses. The data support the classification of H174 as a member of a subfamily of interferon-gamma inducible non-ELR CXC-chemokines. Brain tissues were obtained at autopsy from one patient with AIDS dementia, one patient with multiple sclerosis, and two normal control patients. H174 and Mig were detected by RT-PCR in brain tissue cDNA derived from the patients with pathological conditions associated with activated astrocytes but not in cDNA from control specimens.
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Complejo SIDA Demencia/fisiopatología , Astrocitos/virología , Corteza Cerebral/química , Quimiocinas CXC/genética , Complejo SIDA Demencia/inmunología , Animales , Anticuerpos , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Astrocitoma , Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/virología , Quimiocina CXCL11 , Quimiocinas CXC/análisis , Quimiocinas CXC/inmunología , Quimiotaxis/inmunología , Clonación Molecular , Cricetinae , Cricetulus , Cartilla de ADN , ADN Complementario , ADN Viral/análisis , Femenino , Feto/química , Feto/citología , Expresión Génica , Células HL-60 , Humanos , Interferón gamma/farmacología , Leucocitos/inmunología , Leucocitos/virología , Datos de Secuencia Molecular , Esclerosis Múltiple/inmunología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Células U937 , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.
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ADN Complementario/aislamiento & purificación , Vectores Genéticos , Señales de Clasificación de Proteína , Proteínas/metabolismo , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Quimiocinas/genética , Glicósido Hidrolasas/genética , Humanos , Interferón gamma/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Selección Genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , beta-FructofuranosidasaRESUMEN
Thymus-derived chemotactic agent 4 (TCA4), a new member of the beta-chemokine family, was cloned from a mouse thymic cDNA library. High levels of TCA4 mRNA are expressed in thymus; lower levels of message are found in spleen, heart, and kidney. Anti-TCA4 antibodies were used to localize sites of TCA4 expression within lymphoid tissues. In the thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained with anti-TCA4. TCA4 was also expressed as a meshlike network in splenic white pulp and in the medullary region of the lymph nodes. In addition, some lymph node and splenic blood vessels stained with anti-TCA4 antibodies. Rel B NFkappaB-deficient mice lack a transcription factor required for the generation of dendritic cells and the development of an organized thymic medulla. Rel B-deficient animals express very low levels of TCA4 in the thymus and little or no TCA4 in the periphery. At subnanomolar concentrations, TCA4 is a chemoattractant of mature T cells; the potential role of this novel chemokine in facilitating normal lymphocyte traffic is discussed. TCA4 is also a chemoattractant of cultured mesangial cells. Neutralizing anti-TCA4 mAb was used to demonstrate the specificity of TCA4-mediated cell migration. Finally, competitive binding studies with a SV40-transformed mouse mesangial cell line demonstrated that other murine beta-chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and thymus-derived chemotactic agent 3) do not compete for TCA4 binding.
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Quimiocinas CC/metabolismo , Mesangio Glomerular/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Quimiocinas CC/genética , Quimiocinas CC/inmunología , Quimiotaxis , Clonación Molecular , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Timo/metabolismoRESUMEN
A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.
Asunto(s)
Escherichia coli/metabolismo , Histidina , Mutación Puntual , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Quelantes , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Tiorredoxinas/aislamiento & purificaciónRESUMEN
We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.
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Enteropeptidasa/genética , Fragmentos de Péptidos/biosíntesis , Animales , Fusión Artificial Génica , Sitios de Unión , Catálisis , Bovinos , Enteropeptidasa/biosíntesis , Escherichia coli , Vectores Genéticos , Histidina , Péptidos/metabolismo , Pichia , Proteínas Recombinantes/biosíntesis , Solubilidad , Tiorredoxinas/biosíntesisRESUMEN
Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.