ObjectiveToinvestigatetheplasmidgenechangesinquinolone‐sensitivePseudomonasaeruginosa.Methods 31iso‐lates from January 2011 to December 2013 from various qualified clinical samples in the hospital were collected .In the 31 isolateds , 16 isolates proved sensitive to quinolones by using K‐B method were used as research objects in the study .The isolates growing ou‐side the sensitive ring of ciprofloxacin paper were selected to continuously transferred into other culture dishes until the resistance to quinolones were acquired .Plasmid transformation and extraction were performed on those isolates to confirm the existence of drug‐resistanceplasmidsacquired,andthroughPCRandgenesequenceanalysistodeterminethetypeofplasmids.Results 2iso‐lates of quinolone‐sensitive Pseudomonas aeruginosa acquired drug‐resistance plasmids qnrS and were resistant quinolones induced by continuous transferring for 9 times .Conclusion If antibiotics of inhibitory concentration were often used for the treatment of Pseudomonas aeruginosa infection ,drug‐resistance plasmids were acquired easily .

Adolescent ; Biopsy ; Cystic Adenomatoid Malformation of Lung, Congenital ; diagnosis ; diagnostic imaging ; therapy ; Diagnosis, Differential ; Diagnostic Errors ; adverse effects ; prevention & control ; Humans ; Infant ; Lung Diseases ; diagnosis ; diagnostic imaging ; therapy ; Male ; Middle Lobe Syndrome ; diagnosis ; diagnostic imaging ; surgery ; Tomography, X-Ray Computed ; Treatment Outcome

OBJECTIVETo explore the relationship between body mass index, waist circumference and blood pressure among residents in Chongqing area.

METHODSA total of 5246 residents aged 15 and over in Chongqing area were enrolled in this study by use of stratified sampling and cluster sampling methods. Data on blood pressure (SBP, DBP), pulse, height, body weight, waist and hip circumferences as well as questionnaire survey were analyzed.

RESULTSThe level of SBP and DBP and hypertension prevalence rate were significantly positively correlated with BMI (all P < 0.01). SBP, DBP levels and hypertension prevalence rate were significantly higher in people with abdomen obesity than people with normal waist circumference (all P < 0.01). BMI, waist circumference in hypertensive residents were significantly higher than non-hypertensive residents (all P < 0.01).

CONCLUSIONBlood pressure level and hypertension prevalence rate were closely related with BMI and waist circumference among residents in Chongqing area.

Adolescent ; Adult ; Aged ; Blood Pressure ; Body Mass Index ; China ; epidemiology ; Female ; Heart Rate ; Humans ; Hypertension ; epidemiology ; physiopathology ; Male ; Middle Aged ; Prevalence ; Risk Factors ; Rural Population ; Urban Population ; Waist Circumference ; Waist-Hip Ratio ; Young Adult

OBJECTIVETo investigate the relationship of virological breakthrough and production of neutralizing anti-interferon antibody (NAb) in chronic hepatitis B patients treated with recombinant interferon-alpha (rIFN-alpha).

METHODFour hundred eighty-five patients with histological proven chronic hepatitis B were treated with 5 MU recombinant interferon-alpha 1b (rIFN-alpha1b) thrice weekly for 6-37 months (median 10). Serum HBV DNA, HBeAg and NAb levels of the patients were detected by fluorescent-quantitative PCR, enzymoimmunoassay and antiviral neutralizing biological assay respectively during the therapy.

RESULTSVirological breakthrough occurred in 66 patients (13.6%), and NAb was found in 98 patients (20.2%) of the total 485 patients. The rate of NAb positivity was higher in patients with viral breakthrough than those without it (68.2%, 45/66, vs 12.6%, 53/419, chi(2)=109.06, P < 0.01), and viral breakthrough occurred more in patients with positive NAb than with negative NAb (45.9%, 45/98, vs 5.4%, 21/387, chi(2)=109.06, P < 0.01). The time of the viral breakthrough occurrence and the time of NAb production had a significant correlation (P < 0.01). The occurrence of viral breakthrough was also influenced by the age of patients (P < 0.05) and HBeAg status (P < 0.01) before they were treated.

CONCLUSIONViral breakthrough occurred in 13.6% of our 485 chronic hepatitis B patients treated with recombinant interferon-alpha. Their viral breakthrough and production of NAb production had a significant correlation.

Adult ; Antibodies, Neutralizing ; biosynthesis ; Female ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon Type I ; therapeutic use ; Male ; Recombinant Proteins ; Young Adult

BACKGROUNDIt is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.

METHODSThe HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.

RESULTSThe T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.

CONCLUSIONSThere are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Chloramphenicol O-Acetyltransferase ; metabolism ; Genes, Regulator ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; pharmacology ; Plasmids ; Point Mutation ; Tumor Necrosis Factor-alpha ; pharmacology

Hepatitis B Core Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; virology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.
Bioreactors ; Cloning, Molecular ; Fermentation ; Humans ; Kringles ; genetics ; Pichia ; genetics ; Plasmids ; Plasminogen ; genetics ; isolation & purification ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Angiogenesis Inhibitors ; metabolism ; Autoantigens ; genetics ; metabolism ; Cell Proliferation ; Cells, Cultured ; Collagen Type IV ; genetics ; metabolism ; DNA, Complementary ; genetics ; Electroporation ; Endothelial Cells ; cytology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo study the mechanism of hepatitis B virus infected patients who is negative for HbsAg.

METHODSDNA sequences of 46 patients were analyzed. In these patients, HBsAg was negative but HBV DNA was positive and six new HBsAg variants were identified. Four of the six variants were combined point mutants and two were insertion variants. These S genes were subcloned into eukaryotic expression vector EBO-plpp, and the recombinant eukaryotic expression plasmids were transfected into COS7 cells. Cell lines expressing mutant type HBsAg were obtained. The supernatants were detected by ELISA and RIA.

RESULTSOnly the two-amino acid-insertion variants could be detected and the others failed to react with polyclonal and monoclonal antibodies against HbsAg.

CONCLUSIONThe results indicated that the point mutations and insertions may result in a conformational change of the S gene, which affect HBsAg antigenicity, suggesting a possible relationship between the variants and the negative conversion of HBsAg of the patients.

Animals ; Antigenic Variation ; COS Cells ; Cercopithecus aethiops ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; virology ; Humans ; Plasmids ; genetics ; Point Mutation ; Transfection

OBJECTIVETo investigate the prevalence of three lamivudine-resistant HBV mutants in lamivudine-treated Chinese patients.

METHODSUsing three pairs of of HBV polymerase gene B and C domain fragments were amplified by PCR. The PCR products were then digested by restriction enzyme Nde I and Nla III. The digested products were analyzed by electrophoresis. With this method, the prevalence of the three lamivudine-resistant mutants in lamivudine-treated Chinese patients was investigated.

RESULTSAfter Nde I digestion of p24 and p29 amplified product, HBV wild type could be easily separated from YMDD mutant. At the same time, YIDD could be separated from YVDD mutant after Nla III digestion of p24 and p29 amplified product. By this method, the authors found that these eleven patients were infected with lamivudine-resistant mutants. Six of them were infected with M5501 mutant; five were infected with M550V mutant (one of them had both M550V and L526M mutations).

CONCLUSIONThe method of the present study was demonstrated to be an easy way to detect HBV lamivudine-resistant mutants and can be applied to clinical monitoring of lamivudine resistance.

Adolescent ; Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA Mutational Analysis ; DNA Primers ; genetics ; DNA, Viral ; analysis ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B ; drug therapy ; virology ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Point Mutation ; Polymerase Chain Reaction