RESUMEN
OBJECTIVE: To examine the expression of ADAMTS-7 during the progression of osteoarthritis (OA), defining its role in the pathogenesis of OA, and elucidating the molecular events involved. METHODS: ADAMTS-7 expression in cartilage of a rat OA model was assayed using immunohistochemistry. Cartilage-specific ADAMTS-7 transgenic mice and ADAMTS-7 small interfering (si)RNA knockdown mice were generated and used to analyse OA progression in both spontaneous and surgically induced OA models. Cartilage degradation and OA was evaluated using Safranin-O staining, immunohistochemistry, ELISA and western blotting. In addition, mRNA expression of tumour necrosis factor (TNF)-α and metalloproteinases known to be involved in cartilage degeneration in OA was analysed. Furthermore, the transactivation of ADAMTS-7 by TNF-α and its downstream NF-κB signalling was measured using reporter gene assay. RESULTS: ADAMTS-7 expression was elevated during disease progression in the surgically induced rat OA model. Targeted overexpression of ADAMTS-7 in chondrocytes led to chondrodysplasia characterised by short-limbed dwarfism and a delay in endochondral ossification in 'young mice' and a spontaneous OA-like phenotype in 'aged' mice. In addition, overexpression of ADAMTS-7 led to exaggerated breakdown of cartilage and accelerated OA progression, while knockdown of ADAMTS-7 attenuated degradation of cartilage matrix and protected against OA development, in surgically induced OA models. ADAMTS-7 upregulated TNF-α and metalloproteinases associated with OA; in addition, TNF-α induced ADAMTS-7 through NF-κB signalling. CONCLUSIONS: ADAMTS-7 and TNF-α form a positive feedback loop in the regulation of cartilage degradation and OA progression, making them potential molecular targets for prevention and treatment of joint degenerative diseases, including OA.
Asunto(s)
Proteínas ADAM/inmunología , Retroalimentación Fisiológica , Osteoartritis/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS7 , Envejecimiento/inmunología , Animales , Cartílago/citología , Cartílago/inmunología , Cartílago/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/inmunología , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/inmunología , FN-kappa B/metabolismo , Osteoartritis/metabolismo , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling, and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Anciano , Animales , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/patología , Artritis Experimental/fisiopatología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Femenino , Granulinas , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Ligandos , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Progranulinas , Dominios y Motivos de Interacción de Proteínas , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/fisiología , Adulto JovenRESUMEN
BACKGROUND: To establish a convenient system for the study of human papillomavirus (HPV), we inserted a Saccharomyces cerevisiae selectable marker, Ura, into HPV58 genome and transformed it into yeast. RESULTS: HPV58 genome could replicate extrachromosomally in yeast, with transcription of its early and late genes. However, with mutation of the viral E2 gene, HPV58 genome lost its mitotic stability, and the transcription levels of E6 and E7 genes were upregulated. CONCLUSIONS: E2 protein could participate in viral genome maintenance, replication and transcription regulation. This yeast model could be used for the study of certain aspects of HPV life cycle.
Asunto(s)
Papillomaviridae/fisiología , Saccharomyces cerevisiae/virología , Transcripción Genética , Virología/métodos , Replicación Viral , Perfilación de la Expresión Génica , Genes Virales , Humanos , Transformación GenéticaRESUMEN
Chlamydia trachomatis, an obligate intracellular bacterial pathogen, is the major cause of sexually transmitted diseases worldwide. Although a variety of strategies have been taken to promote the development of a protective vaccine, no ideal vaccine has been generated so far. In this study, we transfected dendritic cells (DCs) with recombinant adenovirus carrying C. trachomatis serovar E major outer membrane protein gene (Ad-MOMP), and investigated their ability to induce specific protection against genital tract chlamydial challenge infection. The results showed that when DCs were transfected with Ad-MOMP in vitro, the DCs exhibited increased expression of CD80 and MHC-II molecules as well as enhanced IL-12 secretion and were able to stimulate T-cell proliferation. The level of IFN-gamma secreted by stimulated T cells was also up-regulated significantly. When the Ad-MOMP transfected DCs were adoptively transferred intravenously to naive mice, they generated Th1-biased cytokine production and mucosal IgA responses specific for C. trachomatis. More importantly, the mice immunized with Ad-MOMP-DC mounted protection against genital tract challenge infection, shown by lower body mass loss, lower chlamydial loads, and less severe pathological changes. In conclusion, Ad-MOMP transfected DCs are capable of inducing effective protective immune responses against C. trachomatis genital infection.
Asunto(s)
Adenoviridae/genética , Infecciones por Chlamydia/terapia , Células Dendríticas/fisiología , Enfermedades de los Genitales Femeninos/terapia , Inmunoterapia Adoptiva/métodos , Porinas/genética , Animales , Células Cultivadas , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Femenino , Enfermedades de los Genitales Femeninos/genética , Enfermedades de los Genitales Femeninos/inmunología , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Ratones , Ratones Endogámicos BALB C , Porinas/inmunología , Transfección , TransgenesRESUMEN
OBJECTIVE: To establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum. METHODS: Two DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA. RESULTS: The standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively. CONCLUSION: The Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.
Asunto(s)
ADN Viral/sangre , Parvovirus B19 Humano/genética , Reacción en Cadena de la Polimerasa/métodos , HumanosRESUMEN
ADAMTS-7, a metalloproteinase that belongs to ADAMTS family, is important for the degradation of cartilage extracellular matrix proteins in arthritis. Herein we report that ADAMTS-7 is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. ADAMTS-7 potently inhibits chondrocyte differentiation and endochondral bone formation, and this inhibition depends on its proteolytic activity. The cysteine-rich domain of ADAMTS-7 is required for its interaction with the extracellular matrix, and the C-terminal four-thrombospondin motifs are necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-7 is an important target of canonical PTHrP signaling, since (i) PTHrP induces ADAMTS-7, (ii) ADAMTS-7 is downregulated in PTHrP null mutant (PTHrP-/-) growth plate chondrocytes, and (iii) blockage of ADAMTS-7 almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and endochondral bone growth. ADAMTS-7 associates with granulin-epithelin precursor (GEP), an autocrine growth factor that has been implicated in tissue regeneration, tumorigenesis, and inflammation. In addition, ADAMTS-7 acts as a new GEP convertase and neutralizes GEP-stimulated endochondral bone formation. Collectively, these findings demonstrate that ADAMTS-7, a direct target of PTHrP signaling, negatively regulates endochondral bone formation by associating with and inactivating GEP chondrogenic growth factor.
Asunto(s)
Proteínas ADAM/metabolismo , Huesos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS7 , Animales , Western Blotting , Desarrollo Óseo , Huesos/embriología , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Condrocitos/citología , Condrocitos/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Noqueados , Proteína Relacionada con la Hormona Paratiroidea/genética , Progranulinas , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de Tejidos , Técnicas del Sistema de Dos HíbridosRESUMEN
Id proteins play important roles in osteogenic differentiation; however, the molecular mechanism remains unknown. In this study, we established that inhibitor of differentiation (Id) proteins, including Id1, Id2, and Id3, associate with core binding factor alpha-1 (Cbfa1) to cause diminished transcription of the alkaline phosphatase (ALP) and osteocalcin (OCL) gene, leading to less ALP activity and osteocalcin (OCL) production. Id acts by inhibiting the sequence-specific binding of Cbfa1 to DNA and by decreasing the expression of Cbfa1 in cells undergoing osteogenic differentiation. p204, an interferon-inducible protein that interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-induced ALP activity and OCL production. We show that 1) p204 disturbed the binding of Id2 to Cbfa1 and enabled Cbfa1 to bind to the promoters of its target genes and 2) that p204 promoted the translocation from nucleus to the cytoplasm and accelerated the degradation of Id2 by ubiquitin-proteasome pathway during osteogenesis. Nucleus export signal (NES) of p204 is required for the p204-enhanced cytoplasmic translocation and degradation of Id2, because a p204 mutant lacking NES lost these activities. Together, Cbfa1, p204, and Id proteins form a regulatory circuit and act in concert to regulate osteoblast differentiation.
Asunto(s)
Diferenciación Celular , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Inhibidoras de la Diferenciación/química , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas Nucleares/metabolismo , Osteogénesis , Fosfoproteínas/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Señales de Exportación Nuclear , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Ubiquitina/metabolismoRESUMEN
Although human papillomavirus (HPV) infections are the primary cause of cervical cancer, the molecular mechanism by which HPV induces cervical cancer remains largely unclear. We used two-dimensional electrophoresis with mass spectrometry to study protein expression profiling between HPV16-positive cervical mucosa epithelial H8 cells and cervical cancer Caski cells to identify 18 differentially expressed proteins. Among them, retinoblastoma-binding protein 4 (RbAp48) was selected, and its differentiation expression was verified with both additional cervical cancer-derived cell lines and human tissues of cervical intraepithelial neoplasia and cervical cancer. Suppression of RbAp48 using small interfering RNA approach in H8 cells significantly stimulated cell proliferation and colony formation and inhibited senescence-like phenotype. Remarkably, H8 cells acquired transforming activity if RpAp48 was suppressed, because H8 cells stably transfected with RbAp48 small interfering RNA led to tumor formation in nude mice. In addition, overexpression of RbAp48 significantly inhibited cell growth and tumor formation. This RbAp48-mediated transformation of HPV16 is probably because of the regulation by RbAp48 of tumor suppressors retinoblastoma and p53, apoptosis-related enzymes caspase-3 and caspase-8, and oncogenic genes, including E6, E7, cyclin D1 (CCND1), and c-MYC. In brief, RbAp48, previously unknown in cervical carcinogenesis, was isolated in a global screen and identified as a critical mediator controlling the transforming activity of HPV16 in cervical cancer.
Asunto(s)
Proteínas Portadoras/metabolismo , Transformación Celular Viral , Regulación Neoplásica de la Expresión Génica , Papillomavirus Humano 16/metabolismo , Proteínas Nucleares/metabolismo , Displasia del Cuello del Útero/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Caspasa 3/biosíntesis , Caspasa 3/genética , Caspasa 8/biosíntesis , Caspasa 8/genética , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Viral/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ciclina D , Ciclinas/biosíntesis , Ciclinas/genética , Electroforesis en Gel Bidimensional , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Papillomavirus Humano 16/genética , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Fenotipo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño/farmacología , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteína de Retinoblastoma/biosíntesis , Proteína de Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/genética , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virologíaRESUMEN
Chlamydia trachomatis is a kind of obligate intracellular bacterial pathogen that causes ocular and sexually transmitted diseases. In this study, we analyzed the codon usage patterns of the C. trachomatis mouse pneumonitis biovar (MoPn) and Homo sapiens. We found large differences between MoPn and human codon usages. To enhance the expression of Chlamydia protein in mammalian cells, the DNA sequence encoding the major outer-membrane protein (MOMP) of MoPn was modified to substitute the human-preferred codons for rarely used codons. The huma-optimized MOMP gene was synthesized and cloned into the pcDNA3 vector, as was the wild-type MOMP gene. The protein expression levels of the human-optimized MOMP and wild-type MOMP genes were compared. The experiments showed that the human-optimized MOMP gene produced significantly higher levels of MOMP protein than the wild-type MOMP, both in vitro and in vivo, but no obvious difference was observed in the levels of modified and native MOMP mRNA expression. The immunogenicity of the 2 constructs was examined using BALB/c mice following intramuscular immunization. The results showed that the mice immunized with the human-optimized MOMP produced higher levels of antigen-specific IgG antibody and showed stronger delayed-type hypersensitivity reactions and proliferative T cell responses than those immunized with the wild-type MOMP. Antigen-specific stimulation of spleen cells obtained from human MOMP DNA immunized mice produced higher levels of interferon-gamma than those obtained from wild-type MOMP DNA immunized mice. Taken together, the data show that human-optimized codon optimization can significantly enhance the gene expression and immunogenicity of the C. trachomatis MOMP DNA vaccine.
Asunto(s)
Formación de Anticuerpos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Formación de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos/genética , Especificidad de Anticuerpos/inmunología , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/prevención & control , Codón/genética , Codón/inmunología , Femenino , Genes Bacterianos/inmunología , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/genética , Vacunas de ADN/farmacocinéticaRESUMEN
Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor alpha1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Proteínas Nucleares/fisiología , Osteogénesis/fisiología , Fosfoproteínas/fisiología , Proteína de Retinoblastoma/fisiología , Animales , Secuencia de Bases , Diferenciación Celular/fisiología , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Cartilla de ADN , Silenciador del Gen , Inmunoprecipitación , Ratones , Proteínas Nucleares/metabolismo , Osteoblastos/citología , Fosfoproteínas/metabolismo , ARN Interferente Pequeño , Proteína de Retinoblastoma/metabolismoRESUMEN
5-Fluorouracil is the first choice chemotherapeutic drug for patients with gastric cancer, but the mechanism that 5-fluorouracil plays the anti-tumor role remains unclear. The aim of this study was to clarify correlated [corrected] proteins induced by 5-fluorouracil in the apoptosis-initiation of human gastric cancer (MGC-803) cells. The time point of apoptosis-initiation induced by 5-fluorouracil in MGC-803 cells was determinated using 5-fluorouracil-withdrawal. Two-dimensional electrophoreses (2-DE) were employed to compare the differentials of protein expressions of the MGC-803 cells at the apoptosis-initiation phase and those of the MGC-803 cells untreated with 5-fluorouracil. The differential proteins included 14 upregulated proteins and 8 downregulated proteins. They indicated a more-than-doubled alteration. These proteins were digested in gels by trypsin and the mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting (PMF) were searched out using the internet available database mascot (http://www.matrixscience.com). The results showed that proteomics analyses have evidenced that many kinds of proteins are involved in the apoptosis initiation of human gastric cancer MGC-803 cells. These proteins are related to metabolism, oxidation, cytoskeleton and signal transduction and other aspects of cells. In conclusion, the experiment model of apoptosis-initiation of human gastric cancer MGC-803 cells induced by 5-fluorouracil based on proteomic analysis has been established, giving an impetus to researches of the mechanism of apoptosis in human gastric cancer, and laying a foundation for the selection of potential drug precursors specific for inducing apoptosis-initiation in human gastric cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Fluorouracilo/farmacología , Proteómica , Neoplasias Gástricas/fisiopatología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/fisiopatología , Línea Celular Tumoral , Fluorouracilo/uso terapéutico , Humanos , Neoplasias Gástricas/tratamiento farmacológicoAsunto(s)
Hepatitis B/inmunología , Antígenos de Histocompatibilidad Clase II/sangre , Antígenos de Histocompatibilidad Clase I/sangre , Linfocitos/inmunología , Línea Celular , Citotoxicidad Inmunológica , Humanos , Células Asesinas Activadas por Linfocinas/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BACKGROUND: To investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation. METHODS: The Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay. RESULTS: The Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro. CONCLUSION: Yeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/ultraestructura , Benzotiazoles , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Cinética , Microscopía Electrónica , Factores de Terminación de Péptidos , Priones/genética , Priones/ultraestructura , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Tiazoles/metabolismoRESUMEN
Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.
Asunto(s)
Resistencia a Múltiples Medicamentos , Leucemia Mieloide Aguda/fisiopatología , Proteómica , Electroforesis en Gel Bidimensional , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Mapeo Peptídico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: To construct the human cytomegalovirus (HCMV) pp65 DNA vaccine vector and VP22 and pp65 coexpressing vector. To evaluate and compare immunological effects in mice. METHODS: Twenty-one BALB/C mice were divided into 3 equal groups: pcDNA3.pp65 group undergoing injection of pcDNA3.pp65 as DNA vaccine, pVP22.pp65 group undergoing injection of pVP22.pp65, and control group undergoing injection of normal saline. HCMV pp65 expression vector pcDNA3.pp65, VP22 and pp65 coexpressing vector pVP22.pp65 were constructed by molecular biology methods. The vectors were immunized into BALB/C mice as DNA vaccines. The T cell proliferation activity and IL-2 biological activity were determined using MTT method. NK activity was detected using LDH release test. The serum IgM and IgG levels of HCMV, the concentrations of IL-2 and IL-4 in serum and supernatant of spleen cells were detected using ELISA method. RESULTS: The HCMV DNA vaccine expression vectors were successfully constructed. Some indexes of the two vaccine groups (pcDNA3.pp65 and pVP22.pp65), that is, T cell proliferation activity (5.11 and 5.55 for SI at 8th week respectively), NK activity (8.74% and 12.08% at 12th week respectively), IgM level (1.20 and 1.58 for A value at 6th week respectively) and IgG level (1.09 and 1.78 for A value at 6th week respectively) were higher than those of negative control, and pVP22.pp65 group was higher than pcDNA3.pp65 group (P < 0.05). The concentrations of IL-2 and IL-4 and the IL-2 biological activity were very low in sera of three groups which showed no significant difference between them (P > 0.05), but higher in the spleen supernatant and the pVP22.pp65 group was highest (411.11 pg/ml, 76.10 pg/ml for the concentrations of IL-2 and IL-4 and 0.22 for IL-2 biological activity). CONCLUSION: The HCMV pp65 could induce certain immunological activity, and VP22 could significantly enhance pp65 in vivo immunological activity.
Asunto(s)
Citomegalovirus/inmunología , Fosfoproteínas/genética , Vacunas de ADN/inmunología , Proteínas de la Matriz Viral/genética , Proteínas Estructurales Virales/genética , Vacunas Virales/inmunología , Animales , Infecciones por Citomegalovirus/prevención & control , Femenino , Vectores Genéticos , Interleucina-2/sangre , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunologíaRESUMEN
Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexually transmitted diseases, and are associated with cardiovascular diseases. The analysis of codon usage may improve our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of target genes to improve their expression for gene therapy. Here, we analyzed the codon usage of C. muridarum, C. trachomatis (here indicating biovar trachoma and LGV), C. pneumoniae, and C. psittaci using the codon usage database and the CUSP (Create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). The results show that the four genomes have similar codon usage patterns, with a strong bias towards the codons with A and T at the third codon position. Compared with Homo sapiens, the four chlamydial species show discordant seven or eight preferred codons. The ENC (effective number of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained by the G+C content, while translational selection and gene length exert relatively weaker influences. Moreover, mutational pressure appears to be the major determinant of the codon usage variation among the chlamydial genes. In addition, we compared the codon preferences of C. trachomatis with those of E. coli, yeast, adenovirus and Homo sapiens. There are 23 codons showing distinct usage differences between C. trachomatis and E. coli, 24 between C. trachomatis and adenovirus, 21 between C. trachomatis and Homo sapiens, but only six codons between C. trachomatis and yeast. Therefore, the yeast system may be more suitable for the expression of chlamydial genes. Finally, we compared the codon preferences of C. trachomatis with those of six eukaryotes, eight prokaryotes and 23 viruses. There is a strong positive correlation between the differences in coding GC content and the variations in codon bias (r=0.905, P<0.001). We conclude that the variation of codon bias between C. trachomatis and other organisms is much less influenced by phylogenetic lineage and primarily determined by the extent of disparities in GC content.
Asunto(s)
Chlamydia/genética , Genes Bacterianos , Animales , Codón , Genes , Genes Fúngicos , Genes Virales , Código Genético , Humanos , Modelos Genéticos , Programas InformáticosRESUMEN
The differentiation of uncommitted mesenchymal cells into osteoblasts is a fundamental molecular event governing both embryonic development and bone repair. The bone morphogenetic proteins (BMPs) are important regulators of this process; they function by binding to cell surface receptors and signaling by means of Smad proteins. Core binding factor alpha-1 (Cbfa1), a member of the runt family of transcription factors, is an essential transcriptional regulator of osteoblast differentiation and bone formation, and this process is positively or negatively regulated by a variety of coactivators and corepressors. We report that p204, an interferon-inducible protein that was previously shown to inhibit cell proliferation and promote the differentiation of myoblasts to myotubes, is a novel regulator in the course of osteogenesis. p204 is expressed in embryonic osteoblasts and hypertrophic chondrocytes in the growth plate as well as in the calvaria osteoblasts of neonatal mice. Its level is increased in the course of the BMP-2-triggered osteoblast differentiation of pluripotent C2C12 cells. This increase is probably due to the activation of the gene encoding 204 (Ifi204) by Smad transcription factor, including Smad1, -4, and -5. Overexpression of p204 enhances the BMP-2-induced osteoblast differentiation in vitro, as revealed by elevated alkaline phosphatase activity and osteocalcin production. p204 acts as a cofactor of Cbfa1: 1) high levels of p204 augment, whereas the lowering of p204 level decreases, the Cbfa1-dependent transcription, and 2) p204 associates with Cbfa1 both in vitro and in vivo. Two nonoverlapping segments in p204 bind to Cbfa1, and the N-terminal 88-amino acid segment of Cbfa1 is required for binding to p204. p204, which is the first interferon-inducible protein found to associate with Cbfa1, functions as a novel regulator of osteoblast differentiation.
Asunto(s)
Interferones/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/fisiología , Osteoblastos/metabolismo , Fosfoproteínas/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Factores de Unión al Sitio Principal , Genes Reporteros , Glutatión Transferasa/metabolismo , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Ratones , Modelos Biológicos , Modelos Genéticos , Osteocalcina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To construct human-SCID chimeric mice through implantation of mononuclear cells from human cord blood and study the immunoreaction of SCID-Hu IC mice immunized with rAd5HPV16L1-E7 vaccine. METHODS: (1) Experiment groups were injected with the suspension of mononuclear cells from human cord blood through a tail vein; the control ones were injected with non serum RPMI 1640 medium. Eight weeks after implantation, blood was collected and human serum IgG level in the mice were tested, and human CD45, CD3 and CD19 were determined. (2) SCID-Hu IC mice were divided into two groups: in group A the mice were immunized intraperitoneally with rAd5HPV16L1-E7 virus and in group B the mice were immunized through nasal drip with rAd5HPV16L1-E7 virus. At the end of fourth week, the serum specific IgG antibody to rAd5HPV16L1-E7 virus, IFN-gamma in culture medium of spleen lymphocyte and T-lymphocyte propagation were tested. RESULTS: (1) In the experiment groups, the number of mice positive for human IgG was 10/15, the average values of CD45, CD3 and CD19 were (9.39+/-4.21), (3.25+/-3.99) and (1.69+/-0.75), respectively. In the control ones, the human IgG, CD45, CD3 and CD19 were negative. (2) The results in the experiment groups showed that the IFN-gamma and T-lymphocyte stimulated by HPV16 protein were higher than those in the non-stimulated group (P less than 0.05). CONCLUSION: (1) The results indicated that the construction of human-SCID chimaera through the implantation of mononuclear cells from human cord blood into SCID mice was successful. They also indicated that the reconstructed SCID-Hu IC mice has the ability to produce immune response against rAd5HPV16L1-E7 recombinant virus.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Fusión Oncogénica/genética , Proteínas Oncogénicas Virales/genética , Vacunas Virales/inmunología , Adenoviridae/genética , Animales , Antígenos CD19/sangre , Complejo CD3/sangre , Femenino , Sangre Fetal/trasplante , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Antígenos Comunes de Leucocito/sangre , Masculino , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/inmunología , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/genética , Recombinación Genética , Linfocitos T/citologíaRESUMEN
OBJECTIVE: To preliminarily evaluate the immunity and safety of the recombinant adenoviruses expressing rotavirus structural proteins VP7 and VP6 in rhesus monkeys to lay a foundation for the development of novel genetic engineering vaccine against rotavirus. METHODS: Baby monkeys were immunized with the recombinant adenoviruses intranasally or orally. Serum IgG against rotavirus was measured with ELISA. During the course of the immunization, besides the daily monitoring of body temperature, weight and clinical symptoms, the routine blood and urine tests and liver and kidney function tests were also conducted. RESULTS: Monkeys immunized via intranasal or oral routes could both generate serum IgG against rotavirus. During the immunization, the temperature of monkeys was normal and body weight raise stably. Both routine blood and urine tests and liver and kidney function tests showed no significant alteration compared with the control group. CONCLUSION: The immunization with the recombinant adenoviruses expressing rotavirus antigens is able to induce rotavirus specific efficient immune responses and is safe to baby rhesus monkeys. The preliminary results implied that the recombinant adenoviruses could be an ideal vaccine for rotavirus and lay a foundation for further studies.
Asunto(s)
Adenoviridae/genética , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Rotavirus/genética , Vacunas Sintéticas , Adenoviridae/metabolismo , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Ingeniería Genética , Macaca mulatta , Rotavirus/inmunología , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/genéticaRESUMEN
OBJECTIVE: To obtain monoclonal antibodies (McAbs) which can be widely used to detect mammalian prions (PrP) and to develop diagnostic tests for screening transmissile spongiform encephalopathies (TSE) as well as for studying pathogenesis of prion-related diseases. METHODS: BALB/c mice were immunized separately with bovine PrP peptide 29-48 (BoP1) and 89-108 (BoP2) coupled to keyhole limpt hemocyan. Two hybridoma cell lines secreting monoclonal antibodies against these peptides were established by cell fusion and 2 to 3 rounds of cell cloning. The reactions of the McAbs to the recombinant bovine (Bo)PrP(25-242), human (Hu)PrP(23-231) and hamster (Ha) PrP (23?231) were tested separately by Western blotting. RESULTS: Through cell fusion, two hybridoma cell lines secreting McAbs against BoP1 and BoP2, designated D11 and D8 accordingly, were identified by ELISA and cell cloning. The McAbs produced by these cell lines reacted well with the recombinant PrP proteins; (Bo) PrP (25-242), (Hu) PrP (23-231), and (Ha) PrP (23-231), respectively. CONCLUSIONS: Two McAbs reacting with bovine, human and hamster PrPs were successfully generated, they are potential to be used to detect PrPs in mammals and to study the mechanism of pathogenesis of TSE.