RESUMEN
Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.
Asunto(s)
Antibacterianos/farmacología , Proteína HMGN2/farmacología , Infecciones por Klebsiella/tratamiento farmacológico , Klebsiella pneumoniae/efectos de los fármacos , Pulmón/efectos de los fármacos , Neumonía Bacteriana/tratamiento farmacológico , Actinas/genética , Actinas/inmunología , Proteínas de Fase Aguda/agonistas , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/inmunología , Animales , Antibacterianos/biosíntesis , Quimiocina CXCL1/agonistas , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Femenino , Expresión Génica , Proteína HMGN2/biosíntesis , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/patología , Klebsiella pneumoniae/fisiología , Lipocalina 2 , Lipocalinas/agonistas , Lipocalinas/genética , Lipocalinas/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas/agonistas , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , ARN Mensajero/genética , ARN Mensajero/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacologíaRESUMEN
High mobility group N (HMGNs) are members of the high mobility group protein family, and are involved in the development and progression of several tumors. HMGN1 and HMGN5 were previously shown to be associated with the bioactivities of osteosarcoma. However, the effects and molecular mechanisms of HMGN2 on osteosarcoma progression remain to be determined. In order to characterize the endogenous expression of HMGN2 in osteosarcoma cell lines, RT-PCR and western blot analysis were performed. Recombinant HMGN2 lentivirus was used to infect the osteosarcoma cell lines with relatively low HMGN2 expression to determine the functional relevance of HMGN2 overexpression in osteosarcoma cell growth and migration in vitro and in vivo, and to investigate the expression levels of Ki-67, PCNA, cyclin D1 and cyclin E. The results showed that osteosarcoma cell proliferation and migration were significantly reduced by HMGN2, as indicated by cell count and wound-healing assays. Cell apoptosis was markedly induced and HMGN2 increased the sensitivity to chemotherapy. When HMGN2 expression was enhanced, the expression of cyclin D1 and PCNA was downregulated in osteosarcoma cells. In addition, the tumor volumes in SaO2 and U2-OS subcutaneous nude mouse models treated with HMGN2 lentivirus were significantly decreased as compared to those of the GFP group. These results suggested that the enhanced expression of HMGN2 in osteosarcoma cells by HMGN2 lentivirus, exerts inhibitory effects on growth and migration of osteosarcoma cells.
Asunto(s)
Neoplasias Óseas/genética , Proteína HMGN2/genética , Osteosarcoma/genética , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Ciclina D1/biosíntesis , Ciclina E/biosíntesis , Progresión de la Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Técnicas de Inactivación de Genes , Células HEK293 , Proteína HMGN2/biosíntesis , Humanos , Antígeno Ki-67/biosíntesis , Lentivirus , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Oncogénicas/biosíntesis , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Trasplante HeterólogoRESUMEN
OBJECTIVE: To construct a prokaryotic expression recombinant for the expression of HMGN2 and to evaluate its antiviral activity against human hepatitis virus. METHODS: The extracellular region cDNA of HMGN2 was isolated and amplified by RT-PCR, and introduced to the prokaryotic expression vector pGEX-4T-1. HMGN2 protein was expressed under IPTG induction and purified by GST protein purification system, then identified by SDS-PAGE and Western blot. The cytotoxicity of fusion HMGN2 to HBV-transfected HepG2. 2.15 cell was evaluated with MTT assay. Different concentration of fusion HMGN2 was applied on the HepG2. 2.15 cell and the cell culture supernatants were harvested after 3 and 6 days treatment. The HBsAg and HBeAg in the supernatants were detected by ELISA and the HBV DNA was detected by RT-PCR. RESULTS: In the range of tested 1-100 microg/mL of HMGN2, no cytotoxicity to HepG2. 2.15 cells was detected by MTT assay. When incubated with HMGN2 at 15 microg/mL for 72 h or 144 h, there was a significant reduction in HBeAg and HBsAg expression as well as the HBV DNA copies. CONCLUSION: pGEX-4T-1/HMGN2 vector was success constructed, and the recombinant HMGN2 protein could inhibit HBV expression and replication in vitro remarkably.
Asunto(s)
Antivirales/farmacología , Escherichia coli/metabolismo , Proteína HMGN2/biosíntesis , Virus de la Hepatitis B/efectos de los fármacos , Proteínas Recombinantes/farmacología , Escherichia coli/genética , Vectores Genéticos/genética , Proteína HMGN2/genética , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Total RNA was extracted from human LAK cell, and a cDNA encoding mature peptide HMG-17 and its alpha helix domain was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1lambdaT-HMG-17 and pGEX-1lambdaT HMG-17alpha helix was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified HMG-17. Analyses of MIC, MEC and MBC indicated that HMG-17 and HMG-17alpha had strong antibacterial activity. MIC of the alpha-helic domain was almost the same as that of HMG17, suggesting that the alpha-helic structure would be essential for the antibacterial activity of HMG-17.
