RESUMEN

BACKGROUND: TCAB1, a.k.a. WRAP53ß or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent. METHODS: The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated ß-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. RESULTS: Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 or increasing the expression of TCAB1 were able to attenuate the cellular senescence process induced by TCAB1 silencing. CONCLUSIONS: This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

RESUMEN

OBJECTIVE: Previous studies have shown that abnormal expression of microRNA-184 leads to a variety of cancers, including pancreatic ductal adenocarcinoma, suggesting microRNA-184 as a new treatment target for pancreatic ductal adenocarcinoma. However, the molecular mechanism of microRNA-184 in pancreatic ductal adenocarcinoma remains unclear. It is important to investigate the effect and role of microRNA-184 in pancreatic ductal adenocarcinoma. METHODS: The clinical and laboratory inspection data of 120 patients with pancreatic cancer admitted to the First Affiliated Hospital of Anhui Medical University were compared. MicroRNA-184 expression in tumor tissues and cells was evaluated using reverse transcription polymerase chain reaction. Flow cytometry and Annexin V/propidium iodide staining were performed to examine cell cycle and apoptosis. Western blotting analysis was conducted to measure the protein expression of p-PI3K, p-AKT, JNK1, C-Myc, C-Jun, caspase-9, and caspase-3. RESULTS: MicroRNA-184 expression was low in patients with pancreatic ductal adenocarcinoma. Survival curve showed that patients with lower expression of microRNA-184 in tumor tissues had a worse prognosis and shorter survival time (P < .05), and the multivariate analysis identified that microRNA-184 was an independent prognostic indicator (P < .05). In vitro studies showed that microRNA-184 overexpression induced apoptosis and suppressed cell cycle transition from G1 to S and G2 phases in pancreatic ductal adenocarcinoma cells. Furthermore, molecular studies revealed that inhibition of microRNA-184 promoted the gene expression of p-PI3K, p-AKT, JNK1, C-Myc, and C-Jun compared with the control group. Overexpression of microRNA-184 led to significantly increased expression of caspase-9 and caspase-3 and significantly decreased expression of Bcl-2. CONCLUSION: This study suggests that microRNA-184 inhibits the proliferation and promotes the apoptosis of pancreatic ductal adenocarcinoma cells by downregulating the expression of C-Myc, C-Jun, and Bcl-2. Our verification of the role of microRNA-184 may provide a novel biomarker for the diagnosis, therapy, and prognosis of pancreatic ductal adenocarcinoma.

Asunto(s)

Apoptosis/genética , Carcinoma Ductal Pancreático/genética , MicroARNs/genética , Neoplasias Pancreáticas/genética , Adulto , Anciano , Biomarcadores de Tumor , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal

RESUMEN

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules.

Asunto(s)

Cápside/metabolismo , Dependovirus/fisiología , Receptores de Superficie Celular/metabolismo , Internalización del Virus , Cápside/ultraestructura , Proteínas de la Cápside/clasificación , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/clasificación , Dependovirus/genética , Glicosilación , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/ultraestructura , Serogrupo , Canales Catiónicos TRPP , Transducción Genética

RESUMEN

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2 Šresolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

Asunto(s)

Streptococcus mutans/enzimología , Transaminasas/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica

RESUMEN

Adeno-associated virus (AAV) is a leading vector for virus-based gene therapy. The receptor for AAV (AAVR; also named KIAA0319L) was recently identified, and the precise characterization of AAV-AAVR recognition is in immediate demand. Taking advantage of a particle-filtering algorithm, we report here the cryo-electron microscopy structure of the AAV2-AAVR complex at 2.8 Å resolution. This structure reveals that of the five Ig-like polycystic kidney disease (PKD) domains in AAVR, PKD2 binds directly to the spike region of the AAV2 capsid adjacent to the icosahedral three-fold axis. Residues in strands B and E, and the BC loop of AAVR PKD2 interact directly with the AAV2 capsid. The interacting residues in the AAV2 capsid are mainly in AAV-featured variable regions. Mutagenesis of the amino acids at the AAV2-AAVR interface reduces binding activity and viral infectivity. Our findings provide insights into the biology of AAV entry with high-resolution details, providing opportunities for the development of new AAV vectors for gene therapy.

Asunto(s)

Cápside/metabolismo , Infecciones por Parvoviridae/virología , Parvovirinae/metabolismo , Receptores de Superficie Celular/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Microscopía por Crioelectrón , Dependovirus , Interacciones Huésped-Parásitos , Humanos , Parvovirinae/genética , Parvovirinae/ultraestructura , Unión Proteica , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/ultraestructura

RESUMEN

Donor organ rejection remains a significant problem. The present study aimed to assess whether transferring a donor's major histocompatibility complex (MHC) genes to the recipient could mitigate rejection in organ transplantation. Seven loci of MHC genes from donor mice were amplified and ligated into vectors; the vectors either contained one K locus, seven loci or were empty (control). The vectors were subsequently injected into the thymus of recipients (in heterotransplants, recipient rats received the vector containing one K locus), following which donor mouse hearts were transplanted. Following the transplantation of allograft and heterograft, electrocardiosignals were viable for a significantly longer duration in recipient mice and rats receiving the donor histocompatibility-2 complex (H-2)d genes compared with those in controls, and in mice that received seven vectors compared with those receiving one vector. Mixed lymphocyte cultures containing cells from these recipients proliferated significantly less compared with mixed lymphocyte cultures containing controls. Also, hearts from H-2d genes-treated recipients demonstrated less lymphocyte infiltration and necrosis compared with the control recipient. The present study concluded that allograft and heterograft rejection may be mitigated by introducing the donor's MHC into the recipient; transferring seven loci has been demonstrated to be more effective than transferring one locus.

RESUMEN

BACKGROUND/AIM: The ELAVL4 (HuD) is a neuron-specific RNA-binding protein expressed in 100% of small cell lung cancer (SCLC) cells and over 50% of neuroblastoma cells. The aim of this study was to investigate the serum HuD concentration in SCLC patients and the possibility of its utilization as a biomarker of small cell lung cancer. MATERIALS AND METHODS: Our study included 47 SCLC cases and 29 normal controls. Indirect competitive inhibition ELISA method was established to detect HuD antigen of serum samples. To design the ELISA system, purified antigen and real positive and negative serum samples were used, and checkerboard titration was performed. The value of current serum biomarkers (Pro-GRP, NSE, CYFRA21-1 and CEA) was obtained from a clinical laboratory. RESULTS: The HuD antigen concentration in the SCLCgroup was significantly higher than that in the normal group (p<0.01). The cut-off value, specificity and sensitivity were 60 ng/ml, 89.7% and 74.5%, respectively. The best linear range was 9.75~600 ng/ml. The sensitivity of the HuD-ELISA assay was much better than the current biomarkers-CEA, NSE and pro-GRP. Also,it was equal to or better than the combined use of two or three indicators. CONCLUSION: The high titres of HuD in SCLC patients and preferable consistency suggested that HuD may serve as a potential diagnostic criterium for SCLC and may serve as a marker of disease progression.

Asunto(s)

Biomarcadores de Tumor , Proteína 4 Similar a ELAV/sangre , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/diagnóstico , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/diagnóstico , Anciano , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Sensibilidad y Especificidad

RESUMEN

Peptide-based therapy, such as modified peptides, has attracted increased attention. IL-17 is a promising therapeutic target for autoimmune diseases, and levels of circulating bioactive IL-17 are associated with rheumatoid arthritis severity. In this study, a modified truncated IL-17RC is generated to ameliorate inflammation and bone destruction in arthritis. The truncated IL-17RC binds to both IL-17A and IL-17F with higher binding capacity compared to nonmodified IL-17RC. In addition, the truncated IL-17RC reduces the secretion of inflammatory and osteoclastogenic factors induced by IL-17A/F in vitro. Moreover, the administration of truncated IL-17RC dramatically improves symptoms of inflammation and inhibited bone destruction in collagen-induced arthritis mice. Collectively, these data demonstrate that modified truncated IL-17RC peptide may be a more effective treatment strategy in the simultaneous inhibition of both IL-17A and IL-17F signaling, whereas the existing agents neutralize IL-17A or IL-17F alone. These suggest that the truncated IL-17RC may be a potential candidate in the treatment of inflammatory associated bone diseases.

Asunto(s)

Artritis Experimental/tratamiento farmacológico , Enfermedades Óseas/tratamiento farmacológico , Interleucina-17/administración & dosificación , Péptidos/administración & dosificación , Sinovitis/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/tratamiento farmacológico , Secuencia de Bases , Huesos/efectos de los fármacos , Línea Celular , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Células RAW 264.7

RESUMEN

Dickkopf-1 (DKK1), a secretory inhibitor of canonical Wnt signaling, plays a critical role in certain bone loss diseases. Studies have shown that serum levels of DKK1 are significantly higher in rheumatoid arthritis (RA) patients and are correlated with the severity of the disease, which indicates the possibility that bone erosion in RA may be inhibited by neutralizing the biological activity of DKK1. In this study, we selected a panel of twelve peptides using the software DNASTAR 7.1 and screened high affinity and immunogenicity epitopes in vitro and in vivo assays. Furthermore, we optimized four B cell epitopes to design a novel DKK1 multiepitope DNA vaccine and evaluated its bone protective effects in collagen-induced arthritis (CIA), a mouse model of RA. High level expression of the designed vaccine was measured in supernatant of COS7 cells. In addition, intramuscular immunization of BALB/c mice with this vaccine was also highly expressed and sufficient to induce the production of long-term IgG, which neutralized natural DKK1 in vivo. Importantly, this vaccine significantly attenuated bone erosion in CIA mice compared with positive control mice. These results provide evidence for the development of a DNA vaccine targeted against DKK1 to attenuate bone erosion.

Asunto(s)

Artritis Experimental/terapia , Resorción Ósea/terapia , Epítopos de Linfocito B , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vacunas de ADN/farmacología , Animales , Artritis Experimental/genética , Artritis Experimental/metabolismo , Artritis Experimental/patología , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Células COS , Chlorocebus aethiops , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/genética

RESUMEN

Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice.

Asunto(s)

Artritis Experimental/metabolismo , Huesos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Osteoblastos/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Compuestos Azo/farmacología , Western Blotting , Densidad Ósea/fisiología , Huesos/citología , Línea Celular , Proliferación Celular/fisiología , Inmunohistoquímica , Masculino , Ratones Endogámicos DBA , Osteoblastos/citología , Dibenzodioxinas Policloradas/farmacología , Pirazoles/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Organismos Libres de Patógenos Específicos , Estadísticas no Paramétricas

RESUMEN

To neutralize the pathological activities of tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL), we engineered and characterized a humanized 8G12 (h8G12) antibody that targeted TNF-α and RANKL. Standard molecular biological and complementarity determining region (CDR)-grafting techniques were used to engineer the h8G12 antibody, and enzyme-linked immunosorbent assays (ELISAs) and Western blotting were employed to determine its binding activation and specificity. TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays were used to evaluate the neutralizing effects of the antibody. The cDNA sequences were established by grafting the murine monoclonal antibody (mAb) 8G12 CDRs into the heavy and light chain (HC and LC) variable regions (VH and VL) of the human mAbs 3DGG_B and 1I9R_L, respectively. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells to produce the h8G12 antibody, which could simultaneously recognize TNF-α and RANKL. In addition, the h8G12 antibody reduced the TNF-α-mediated apoptosis of L929 cells by 25.84%. Furthermore, the h8G12 antibody significantly inhibited leukocyte infiltration in a murine allergic contact inflammation model. Concurrent with the inhibition of apoptosis, the h8G12 antibody significantly reduced the number of osteoclast-like cells in a dose-dependent manner. These results demonstrated that the h8G12 antibody neutralized the activities of TNF-α and RANKL and that it might be a potential candidate for the treatment of inflammatory bone diseases, such as rheumatoid arthritis (RA).

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Osteoclastos/inmunología , Ingeniería de Proteínas/métodos , Ligando RANK/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Monoclonales/genética , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Osteoclastos/efectos de los fármacos , Ligando RANK/genética , Factor de Necrosis Tumoral alfa/genética

RESUMEN

OBJECTIVE: To assess whether the occurrence of coronary artery lesion was correlated with the changes of endothelial progenitor cell (EPC) number and function in murine model of Kawasaki disease (KD). METHODS: Lactobacillus casei cell wall extract (LCWE) was prepared and then C57BL/6 mice received a single intraperitoneal injection of LCWE for inducing KD. Twenty-four mice were categorized randomly into 3 groups: KD model group at Day 14 post-injection, KD model group at Day 56 post-injection and control group with an intraperitoneal injection of phosphate buffered solution (n = 8 each). The number of circulating EPC was defined as CD34(+)Flk-1(+)CD45(-) from mice. Meanwhile, bone marrow mononuclear cells were cultured in vitro to expand EPC for functional analysis. After 7 days of culturing, EPC were inoculated onto culture plate and thiazolyl blue assay was used to measure the absorbance value by enzyme labeling instrument to evaluate the proliferation. The adhesion of EPC was performed by replating cells on fibronectin coated dishes and then counting the number of adherent cells. The migration of EPC was assayed by Transwell. RESULTS: Focal inflammatory infiltrate was evident in coronary artery trunk and a series of branches at Day 14 post-injection. The inflammatory cell infiltrate consisted of mononuclear lymphocytes. The number of circulating EPC were significantly lower in the Day 14 LCWE-treating murine model versus the controls (0.017% ± 0.008% vs 0.028% ± 0.007%, P < 0.01). Disruption of elastin was consistently observed at Day 56 post-injection. And there was no apparent recovery in number of EPC (0.016% ± 0.007%, P < 0.01). When bone marrow mononuclear cells were cultured in vitro, the colony-forming ability of EPC decreased in the KD model group at Day 14 post-injection versus the controls. Test of proliferating ability showed that the absorbance was 0.39 ± 0.11 in MTT experiment and decreased than the controls (0.61 ± 0.14, P < 0.01). Adhesion and migration were also down-regulated versus the controls ((3.1 ± 0.6) and (3.2 ± 0.6) vs (6.4 ± 1.2) and (6.2 ± 0.5) cells/HPF, both P < 0.01). In the KD model group at Day 56 post-injection, the colony-forming ability of EPC was not recovered significantly. Proliferation ability, adhesion and migration were still decreased compared to the controls (0.38 ± 0.09, (3.12 ± 0.56) cells/HPF and (3.29 ± 0.63) cells/HPF, all P < 0.01). CONCLUSION: The occurrence of coronary artery lesion may be correlated with the down-regulation of EPC number and function in murine model of KD.

Asunto(s)

Modelos Animales de Enfermedad , Células Endoteliales/citología , Síndrome Mucocutáneo Linfonodular , Células Madre/citología , Animales , Regulación hacia Abajo , Masculino , Ratones , Ratones Endogámicos C57BL , Síndrome Mucocutáneo Linfonodular/metabolismo , Síndrome Mucocutáneo Linfonodular/patología

RESUMEN

The branched-chain amino-acid aminotransferase from Streptococcus mutans (SmIlvE) was recombinantly expressed in Escherichia coli with high yield. An effective purification protocol was established. A bioactivity assay indicated that SmIlvE had aminotransferase activity. The specific activity of SmIlvE towards amino-acid substrates was found to be as follows (in descending order): Ile > Leu > Val > Trp > Gly. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. The structure of SmIlvE was solved at 1.97 Å resolution by the molecular-replacement method. Comparison with structures of homologous proteins enabled the identification of conserved structural elements that might play a role in substrate binding. Further work is needed to confirm the interaction between SmIlvE and its substrates by determining the structures of their complexes.

Asunto(s)

Streptococcus mutans/enzimología , Transaminasas/química , Secuencia de Aminoácidos , Bioquímica/métodos , Calibración , Biología Computacional/métodos , Cristalización , Escherichia coli/metabolismo , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Difracción de Rayos X

RESUMEN

Targeting tumor necrosis factor-α (TNF-α) and activator of NF-κB ligand (RANKL) has been proved highly successful in rheumatoid arthritis (RA) models and patients. This raises a possibility whether a single agent simultaneously targeting TNF-α and RANKL provides a potential therapeutic opportunity. This study aimed to design a dual functional vaccine and evaluate its therapeutic effects in RA mice model. Standard molecular biological techniques were used to generate human RANKL-TNF-like core fusion protein (RTFP-2) vaccine. High titers of antibodies against human TNF-α and RANKL were elicited and the RTFP-2 antiserum decreased TNF-α mediated apoptosis of L929 cells to 41% compared with 90% in positive controls. In addition, the antiserum completely abrogated osteoclastogenesis in vitro. Immunization with RTFP-2 also reduced the mortality of TNF-α induced cachexia from 56% to 28%. The RANKL-mediated hypercalcemic effects were significantly attenuated in RTFP-2 vaccinated mice. Furthermore, RTFP-2 vaccine significantly mitigated the incidence and severity of CIA via inhibition of inflammation and bone resorption. Our results showed the RTFP-2 vaccine of dual targets ameliorated the symptoms of CIA mice, suggesting the potential possibility to treat inflammatory bone diseases such as RA.

Asunto(s)

Artritis Experimental/prevención & control , Ligando RANK/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de Necrosis Tumoral alfa/uso terapéutico , Vacunas/uso terapéutico , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Artritis Experimental/inmunología , Artritis Experimental/patología , Huesos/inmunología , Huesos/patología , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ligando RANK/química , Ligando RANK/genética , Ligando RANK/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas/química , Vacunas/genética , Vacunas/inmunología

RESUMEN

α-Hemolysin (Hla) is a self-assembling, channel-forming toxin that is secreted by Staphylococcus aureus and is central to the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections in animal models. In this study, we report that baicalin (BAI), a natural compound that lacks anti-S. aureus activity, could inhibit the hemolytic activity of Hla. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that BAI binds to the binding sites of Y148, P151, and F153 in the Hla. This binding interaction inhibits heptamer formation. Furthermore, when added to S. aureus cultures, BAI prevents Hla-mediated human alveolar epithelial (A549) cell injury. In vivo studies further demonstrated that BAI protects mice from S. aureus pneumonia. These findings indicate that BAI hinders the cell lysis activity of Hla through a novel mechanism of interrupting the formation of heptamer, which may lead to the development of novel therapeutics that aim against S. aureus Hla.

Asunto(s)

Antiinfecciosos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Flavonoides/farmacología , Proteínas Hemolisinas/antagonistas & inhibidores , Neumonía Estafilocócica/prevención & control , Staphylococcus aureus/efectos de los fármacos , Animales , Antiinfecciosos/metabolismo , Toxinas Bacterianas/metabolismo , Sitios de Unión , Flavonoides/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Hemolisinas/metabolismo , Hemólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Staphylococcus aureus/metabolismo , Relación Estructura-Actividad

RESUMEN

S-Ribosylhomocysteinase (LuxS) encoded by the luxS gene from Streptococcus mutans plays a crucial role in the quorum-sensing system. LuxS was solubly expressed in Escherichia coli with high yield. The purity of the purified target protein, which was identified by SDS-PAGE and MALDI-TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space group C222(1), with unit-cell parameters a = 55.3, b = 148.7, c = 82.8 Å.

Asunto(s)

Proteínas Bacterianas/química , Liasas de Carbono-Azufre/química , Streptococcus mutans/enzimología , Cristalización , Cristalografía por Rayos X

RESUMEN

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = ß = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

Asunto(s)

Candida/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Urato Oxidasa/química , Urato Oxidasa/metabolismo , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica , Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Urato Oxidasa/genética , Urato Oxidasa/aislamiento & purificación

RESUMEN

Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS-PAGE and MALDI-TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Šresolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å.

Asunto(s)

Proteínas Bacterianas/química , Fosforribosilglicinamida-Formiltransferasa/química , Streptococcus mutans/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Tampones (Química) , Cristalización , Difusión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Fosforribosilglicinamida-Formiltransferasa/genética , Fosforribosilglicinamida-Formiltransferasa/aislamiento & purificación , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Difracción de Rayos X , Rayos X

RESUMEN

BACKGROUND: Donor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor's major histocompatibility complex (MHC) gene to the recipient would mitigate the rejection of transplanted hearts in mice. METHODS: H-2K(k) gene from donor mice was amplified using nested polymerase chain reaction (PCR) and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients' thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy. RESULTS: Southern analysis following nested PCR revealed clear expression of H-2K(k) gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K(k) than in those receiving control cells. Flow cytometric analysis using an anti-H-2K(k) antibody confirmed its expression in H-2K(k) treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K(k) treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K(k) treated mice and large areas of necrosis. CONCLUSION: Rejection of transplanted hearts can be mitigated substantially by introducing the donor's MHC into the recipient.

Asunto(s)

Trasplante de Corazón/inmunología , Trasplante de Corazón/métodos , Complejo Mayor de Histocompatibilidad/genética , Animales , Southern Blotting , Electrocardiografía , Femenino , Citometría de Flujo , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Reacción en Cadena de la Polimerasa

RESUMEN

BACKGROUND: It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined. METHODOLOGY/PRINCIPAL FINDINGS: We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant ß-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0-10.0 and at temperatures ≤ 40 °C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C2-C8), and its suitable substrate was p-nitrophenyl caproate (C6) with optimal catalytic conditions of 39 °C and pH 8.0. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.

Asunto(s)

Esterasas/metabolismo , Mycobacterium tuberculosis/enzimología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Esterasas/química , Esterasas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción