Objective To screen promoter DNA-binding protein of inducible nitric oxide synthase gene by using phage display technique from human liver cDNA library, and to study the expression and regulation mechanism of iNOS gene. Methods The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame(ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase(CAT)activity was determined by enzyme linked immunosorbent assay(ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. Results The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. Conclusion The iNOS gene promoter identified in this study has shown to have transcription activity,and iNOS promoter DNA-binding proteins havs been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.

Abstract?AIM: To analyze the effects of femtosecond laser assisted cataract surgery ( FLACS ) in the treatment of cataract and its effect on prognosis.?METHODS:Forty-two cases (42 eyes) of patients with cataract who were treated in the Department of Ophthalmology in our hospital between January 2012 and December 2014 were selected as the study objects. According to the order of treatment, they were divided into control group and observation group, 21 cases in each. The control group was treated with traditional phacoemulsification cataract surgery ( PCS ) . On the basis, the observation group was treated with femtosecond laser. The effective phacoemulsification time (EPT), cumulative dissipated energy (CDE), fluid flow and monitored pressure of the two groups were recorded.The rate of corneal endothelial loss and the situation of Tyndall phenomenon were statistically analyzed.The two groups were followed up for 1a.The long-term visual acuity recovery was observed.The best corrected visual acuity ( BCVA ) was recorded, and the long-term complications were statistically analyzed.?RESULTS: 1 ) The total response rate in observation group was 95% while in control group was 90% ( P>0.05);2) the surgery time of the observation group was longer than that of the control group ( P<0.05) but EPT was shorter than that of the control group.CDM and liquid flow were less than those of the control group ( P<0.05 ); 3 ) at 1d after surgery, there was no significant difference in intraocular pressure between the two groups (P>0.05); the rates of Tyndall phenomenon and corneal endothelial loss in the observation group were lower than those in the control group ( P<0.05);4) BCVA of the two groups at different time after surgery were significantly higher than that before surgery (P<0.05). However, at 1d, 3mo, 6mo and 1a after surgery, BCVA of the observation group was better than that of the control group ( P <0.05 ); 5 ) the incidence of complications in the observation group after surgery (14%) was lower than that in the control group (43%) (P<0.05).?CONCLUSION: The surgical effects of FLACS in the treatment of cataract are good.After surgery, the visual acuity of patients is improved significantly and the incidence of postoperative complications is low. However, the surgery time is long and cost is high, so it is difficult to popularize.

Animals ; Hexanes ; blood ; toxicity ; Kidney ; drug effects ; pathology ; Liver ; drug effects ; pathology ; Lung ; drug effects ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

Air ; analysis ; Air Pollutants, Occupational ; analysis ; Humans ; Occupational Exposure ; Phthalic Acids ; analysis ; Spectrophotometry ; methods ; Workplace

Objective To screen 18 endophytes from Pseudolarix kaempferi Gord for molluscicidal effect and identify them by morphology. Methods Molluscicidal tests were performed according to the immersion test suggested by WHO and the strain screened was identified by the slide culture. Results The mortality rates of snails immersed by JJ18 broth salified (pH=7) were 26.7%, 76.7% and 100.0% for 24,48 h and 72 h, respectively, and 53.3% and 86.7% in 5% and 10% concentrations of JJ18 broth, respectively. The active components were extracellular moiety of the broth which had no acute toxicity to fish, and JJ18 strain belonged to Aspergillus. Conclusion Extracellular moiety of endophyte JJ18 from Pseudolarix kaempferi Gord is a new resource of molluscicide.

Carotid Artery Diseases ; complications ; Fasciitis, Necrotizing ; complications ; Female ; Humans ; Middle Aged

AIM: To gain cdcSTBX25A-fas chimeric gene bearing the regulative fragment cdcSTBX25A and opening read frame of fas in order to construct and identify eukaryotic expression vectors, pAdTrack-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas, which have the potential to transfer the tumor proliferative signal to promoting-apoptosis signal through up-regulate the fas expression by c-myc. METHODS: A pair of primers were designed according to the known sequences of cdcSTBX25A and fas. The cdcSTBX25A-fas chimeric gene was obtained by PCR reaction and inserted into the two plasmids pAdTrack-CMV and pAdTrack, respectively. The two recombinant plasmids were transferred into E. coli DH5?. The positive clones were screened in LB media with 50 mg/L kanamycin and identified by agarose gel electrophoresis, endonuclease digestion and PCR. The nucleotide sequence of inserted cdcSTBX25A-fas was determined by dideoxy chain termination method. Using software, BLAST was conducted to analyze the structure and sequence of the target fragments and compared with GenBank. RESULTS: The chimeric target gene, cdcSTBX25A-fas, of 1 603 bp was obtained. The positive host bacteria E. coli DH5? of recombinant plasmids were screened and amplified. The double-enzyme digestion showed the pAdTrack-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas presenting 9.2 kb, 1.6 kb bands, and 8.3 kb, 1.6 kb bands respectively. The sequence analysis confirmed that the two shuttle plasmids containing 1 597 bp cdcSTBX25A-fas with the ORF of fas. CONCLUSION: The eukaryotic expression plasmids pAd-Track-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas were successfully constructed.

A fragment sized 400bp of White spot syndrome virus（WS SV，formerly de signated NOSV），recovered from recombinant plasmid pAFD, was labeled with Digox igenin as a probe to detect dynamic distribution of WSSV within 120h and 72h in crawfishes（Cambarus proclarkii） inoculated WSSV by oral taking and injecti on r espectively. Stomach epithelium, intestine epithelium, heart, gill, haemolymph, muscle, hepatopancreas, hypoderm, connective tissue and ovary of infected crawfi shes were examined for WSSV. In both groups, WSSV was first detected in heamoly mph at 12h p.i. and then disappeared. Again it was detected at 96h p.i. only in oral infection group and maintained till 120h p.i., but it didn't appear at 72h p.i. in injection group. WSSV in heart, muscle was detected at 36h p.i. in oral infection group and 24h p.i. in injection group respectively, and then increased generally. In addition, WSSV in intestine epithelium, connective tissue, ovary of oral infection group and intestine epithelium, hypoderm, ovary of injection g roup could also be detected. In dead crawfishes after 120h and 72h p.i. in two groups, WSSV could be detected in all the examined tissues and it demonstrated t hat systemic infection occurred in the animales. The tissue containing more amo unts of WSSV was hypoderm in oral infection group, while intestine epithelium, g ill, hypoderm, ovary in injection infection group. It deduced that WSSV first a ppears in haemolymph and then goes into heart, muscle and other tissues and prol iferates in them. Once again, WSSV is released into heamolymph resulting in syst emic infection till crawfishes' death.

AIM: To investigate the possible mechanism of PRL-2 in invasive metastasis of tumors.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the transfectants were selected by growth in the medium supplemented with G418.Zymographic analysis of metalloproteinases(MMPs) activity was performed,RT-PCR was used to determine the mRNA levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2,the protein levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2 were analyzed by Western blotting.The effects of the special inhibitor of PRL-2 on transfected cells were also observed.RESULTS: The stable cell line selected by G418 was identified by RT-PCR and Western blotting.More abundance of MMP-2,MMP-9 and its activated type were secreted by the CL-1-PRL-2 cells than untransfected cells and transfected vector cells(P

Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity.