ABSTRACT
BACKGROUND:Brahma-related gene 1 (Brg1), a catalytic subunit of an important chromatin remodeling complex, has been considered as a key nuclear transcriptional factor, and tends to be decreased in diabetic cardiomyopathy. OBJECTIVE:To construct an adenovirus vector carrying Brg1, and observe its protective role in oxidative stress induced-cardiomyocyte apoptosis. METHODS:The recombinant adenovirus plasmid was linearized and transfected into HEK293 cel s using Fugene HD for packaging and amplification. The adenovirus particles were further purified, quantified, and sequential y transfected to cardiomyocytes of neonatal Sprague-Dawley rats. The Adeno-EGFP transfected and non-transfected cardiomyocytes were used as control group. 24 hours later, the transfection efficiency was observed by fluorescent microscope, and expressions of Brg1 mRNA and protein were detected by quantified PCR and western blotting. After treatment with 100 μmol/L H2O2 for 12 hours, the expressions of Brg1 protein and cleaved-Caspase 3 were measured by western blotting, and cel apoptosis was analyzed by flow cytometry. RESULTS AND CONCLUSION:(1) The recombinant adenovirus vector of Brg1 had been successful y transfected into cardiomyocytes with higher expressions of Brg1 mRNA and protein, and the transfection efficiency reached more than 90%. (2) After H2O2 treatment, the Brg1 was significantly down-regulated in contrast to the up-regulation of cleaved-Caspase 3;the flow cytometry data showed that the apoptotic cel s were increased. But in Adeno-Brg1 transfected cardiomyocytes, the H2O2 induced cel apoptosis was significantly decreased compared with non-transfected cel s and empty vector transfected cel s. (3) These results suggest that oxidative stress can directly inhibit the Brg1 expression, and overexpression of Brg1 can protect the cardiomyocytes from cel apoptosis induced by oxidative stress.
ABSTRACT
BACKGROUND:Methicil in-resistant Staphylococcus aureus (MRSA) infection had been a global problem up to 1980s, and it has become a leading pathogen giving rise to nosocomial infections now. OBJECTIVE:To determine the molecular types and drug susceptibilities of Staphylococcus aureus prevailed in burn ward, and to provide a basis for preventing and control ing MRSA intections. METHODS:A total of 53 Staphylococcus aureus strains were col ected from the burn ward in the Urumqi General Hospital of Lanzhou Military Region of Chinese PLA. These MRSA strains were identified by PCR and cefoxitin disc diffusion test, and al MRSA strains were typed by spa, SCCmec and MLST typing. In the meanwhile, antibiotic susceptibilities of 17 kinds of drugs, such as oxacil in, to Staphylococcus aureus were also determined, and drug resistance of different types of Staphylococcus aureus especial y MRSA, was analyzed. RESULTS AND CONCLUSION:Among 53 Staphylococcus aureus strains, 43 were identified as MRSA, containing determined for amplification of meoA (n=41) and positive for cefoxitin disc diffusion test (n=2). Three SCCmec types, four spa types, and three ST types were found. The major predominant clone was ST239-MRSA-III-t030 (90.7%), with highest resistant to oxacil in and other nine antibiotics. In conclusion, the higher MRSA isolation rate from the burn ward, and ST239-MRSA-III-t030, as the predominant clone, presents with an outbreak in the burn ward and stronger resistance to many different families of antibiotics.
ABSTRACT
BACKGROUND:Methicil in-resistant Staphylococcus aureus has been a primary pathogen of nosocomial infections worldwide. To construct a quick and easy knockout method is an important technique of studying virulence and resistance of methicil in-resistant Staphylococcus aureus. OBJECTIVE:To construct the Staphylococcus aureus gene knockout plasmid for understanding the antibiotic resistance and virulence of Staphylococcus aureus. METHODS:pUC19 was considered as a basic skeleton of construction. pLE194Ts temperature-sensitive replicon and tetracycline resistance gene fragment pHY300PLK plasmid in pCL52.1 were bound to EcoR I site in pUC19 by high assurance amplification. Al multiple clone sites in pUC19 were reserved. The Escherichia coli-Staphylococcus aureus shuttle plasmid was obtained. The N315 dapB gene knockout plasmid was obtained through gene knockout technology. This strain was eventual y identified by multiplex-PCR. RESULTS AND CONCLUSION:The Escherichia coli-Staphylococcus aureus shuttle plasmid, pYZ1 and pYZ8, was successful y constructed, and had been used in Staphylococcus aureus gene knockout. Homologous recombinant plasmid pYZ-ΔdapB was constructed by restriction enzyme digestion and overlap technique. After genetical y modification in RN4220, the constructed gene knockout plasmid pYZ-ΔdapB was introduced to N315 to be screened in the low culture temperature. The deletion strain was successful y obtained after being identified by multiplex-PCR. Above data suggested that pYZ1 and pYZ8 can be successful y used for Staphylococcus aureus gene detection, which provides a tool to study resistance and virulence of clinical Staphylococcus aureus strains.