The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.

Objective To construct PPAR?and PPAR?response element (PPRE)-controlled luciferase expression vectors,and to determine whether the traditional Chinese medicine emodin activates PPAR?and improves the glucose uptake by HepG2 hepatocytes.Methods (1) PPAR?and PPRE luciferase expression vectors were constructed and were applied to screen more than 20 ingredients of the traditional Chinese medicine. (2) HepG2 cells were incubated with emodin which can activate PPAR?and PPRE luciferase activity,and the PPAR?mRNA expression level was evaluated by RT-PCR/Southern blot.(3) PPAR?and glucose transporter 2 (Glut2) proteins were determined by Western blot analysis in HepG2 cells treated with emodin.(4) The glucose uptake rate was measured using 2-deoxy-[~3H]-D-glucose in HepG2 cells after treatment with emodin.Results (1) Emodin stimulated luciferase activity controlled by PPRE in dose-dependent manner at concentrations of 0.04 to 180?mol/L in COS-7 cells.The highest value was about 4 folds of control in the cells treated with 90?mol/L emodin (P

OBJECTIVETo explore the value of phase ordering with automatic window selection(PAWS)and simultaneous multiple volume(SMV)algorithm double respiratory navigator-gated two-dimensional(2DNAV)dual inversion recovery(DIR)fast spin echo(FSE)high-resolution black-blood coronary artery wall magnetic resonance imaging(MRI)and evaluate its advantages and limitations.

METHODSPAWS and SMV 2DNAV DIR FSE high-resolution black-blood MRI was performed in 21 healthy volunteers. The images were evaluated qualitatively by using four grades(grade 0can not evaluate;grade 1bad;grade 2good;grade 3perfect). Images defined as grade 0 and grade 1 were excluded and those defined as grade 2 and 3 were evaluated further. Thickness of proximal(or middle)segment of right coronary artery(RCA)and left anterior descending branch(LAD)were measured. The difference of wall thickness was analyzed by using two-tailed independent sample t-test. P values of less than 0.05 were considered statistically significant.

RESULTSAmong the 38 slice images,31 slices(RCA13 slices,LAD18 slices;grade 214 slices,grade 317 slices)were obtained for further evaluation. The mean thickness of RCA and LAD was(0.94±0.16)and(0.89±0.15)mm,respectively,and the difference was not significant(t=-0.790,P>0.05).

CONCLUSIONPAWS and SMV algorithm 2DNAV DIR FSE high-resolution black-blood MRI has certain clinical value for coronary artery wall imaging.

Adult ; Coronary Vessels ; anatomy & histology ; Female ; Humans ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Young Adult

OBJECTIVETo study the role of cerebrovascular hemodynamic indexes (CVHI) changing in stroke and to provide reference for stroke prevention and risk factor study.

METHODSFrom 2003 to 2004, participants aged 40 years and above in two communities in Fengxian district were recruited by cluster sampling. Risk factors of stroke and CVHI were investigated and checked during baseline investigation. A total of 10 565 individuals completed the survey and met the inclusion criterion. After baseline investigation, the cohort was followed up for stroke occurrence. Relative risk (RR) of CVHI and common risk factors were estimated by cohort study design.

RESULTSAge of the cohort was (56.2 ± 11.4) years. 4444 (42.1%) were males and 6121 (57.9%) were females. Total follow-up duration was 67 885.7 person-years. A total of 195 stroke cases occurred and incidence density of stroke was 287.2 per 100 000 person-years. Stroke incidence in exposure groups of hypertension, heart disease and alcohol drinking was 3.47% (108/3118), 2.96% (21/710) and 2.50% (47/1882), respectively. The incidence in corresponding non-exposure group was 1.17% (87/7448), 1.77% (174/9855) and 1.70% (148/8683) respectively. There was significant difference between 2 groups (χ(2) value was 62.72, 4.56 and 4.94, respectively, P < 0.05). Stroke incidence in CVHI score < 25, 25 - 49, 50 - 74 and ≥ 75 groups was 9.12% (59/647), 5.68% (44/775), 2.52% (39/1545) and 0.72% (53/7403)(χ(2)trend = 273.57, P < 0.05), respectively. Incidence of stroke in 40 - 49, 50 - 59, 60 - 69, ≥ 70 years age group was 0.22% (8/3565), 1.28% (43/3357), 2.71% (50/1848) and 5.88% (94/1600) (χ(2)trend = 181.48, P < 0.05), respectively. Multiple Cox regression analysis indicated that RR (95%CI) value of hypertension and cigarette smoking was 1.40(1.02 - 1.92) and 1.59(1.19 - 2.12), respectively when comparing with non-exposure group. RR (95%CI) value in CVHI score < 25, 25 - 49 and 50 - 74 points group were 6.15 (4.08 - 9.26), 4.55 (2.98 - 6.96) and 2.68 (1.75 - 4.09), respectively when comparing with the score ≥ 75 points group. RR (95%CI) value in age 50 - 59, 60 - 69 and ≥ 70 years group was 4.61 (2.16 - 9.82), 7.81 (3.67 - 16.60) and 13.49(6.44 - 28.24), respectively when comparing with below 40 years group.

CONCLUSIONCVHI score is the strong independent predictive factor and hypertension, cigarette smoking and age are the independent risk factors of stroke.

Aged ; Brain ; physiopathology ; Cohort Studies ; Female ; Hemodynamics ; Humans ; Male ; Middle Aged ; Risk Factors ; Stroke ; epidemiology ; etiology ; physiopathology

Leptin, which is a plasma protein produced by the obese gene, is expressed and secreted by adipocytes. The clearance of lepdn from the circulation is unknown. But, markedly elevated serum leptin concentrations have recently been reported in patients with chronic renal failure. The purpose of the present study was to investigate plasma leptin concentration of patients with chronic renal failure and evaluate the factors affecting plasma leptin levels. Plasma leptin, insulin, and body mass index were determined in 34 patients with chronic renal failure and 55 control subjects. The plasrna leptin concentrations were not significantly different between patients with chronic renal failure and control subjects (9.4+/-11.8 vs 4.9+/-4.2ng/ml, P>0.05). The serum leptin concentrations were not significantly higher in both male and female CRF patients compared with control subjects (3.96+/-5.72 vs 2.48+/-1.65, P=0.1947, 17.07+/-14.02 vs 7.49+/-4.63ng/ml, P=0.07, respectively). And, there was no significant correlation between serum creatinine and plasma leptin. However, there was significant correlation between plasma leptin concentration and insulin level (P<0.05). We fit a multiple linear regre- ssion analysis with plasma leptin level as the dependent variable in CRF. Sex (male vs female) (P< 0.001) and insulin (P=0.004) were independently associated with plasma leptin level in CRF. These results suggested that plasma leptin level was regulated or affected by multiple factors inclu- ding sex and insulin resistance. Additional study is required to evaluate relationship between plasma leptin and insulin resistance in chronic renal failure.
Adipocytes ; Body Mass Index ; Creatinine ; Female ; Humans ; Insulin ; Insulin Resistance ; Kidney Failure, Chronic* ; Leptin* ; Male ; Plasma*

OBJECTIVETo realize quadratic formula optimization of Renshen Jianxin Capsule (RJC) by screening Chinese herbs with major anti-myocardial ischemia effect in RJC and optimize their optimal dosages.

METHODSBy following "uniform design-pharmacodynamic experiment-mathematical modeling-formula optimization", authors employed U10(10(8)) uniform design in the experiment. Eight Chinese herbs contained in RJC were taken as observatory factors. Electrocardiograph (ECG) changes of myocardial ischemia induced by isoproterenol were taken as pharmacodynamic indices. The mathematical model between herbal factors and pharmacodynamic indices was established using stepwise regression analysis to screen Chinese herbs with major anti-myocardial ischemia effect. Their optimal dosages were optimized using the grid algorithm.

RESULTSThe regression equation was y =1. 7889 -0. 3247 Ginseng xSalvia Miltiorrhiza -0. 0663 Astragalus membranaceus xOriental Waterplantain tuber. Forecasting factors included were Ginseng, Salvia Miltiorrhiza, Astragalus membranaceus, and Oriental Waterplantain tuber. The optimal formula dosage calculated by the grid algorithm was Ginseng 1. 62 g, Astragalus membranaceus 4. 62 g, Salvia Miltiorrhiza 2. 43 g, and Oriental Waterplantain tuber 1. 66 g.

CONCLUSIONUniform design combined with stepwise regression analysis and grid algorithm were able to realize quadratic formula optimization of RJC.

Astragalus membranaceus ; Chemistry, Pharmaceutical ; standards ; Coronary Artery Disease ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Electrocardiography ; Humans ; Isoproterenol ; Myocardial Ischemia ; drug therapy ; Panax ; Salvia miltiorrhiza

Objective To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes(AMEs)genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital,Huzhou district,Zhejiang province,China.Methods 40 strains of Enterobacter cloacae were isolated from the inpatients between September,2003 and November,2004.5 kinds of 16S rRNA methylase gene (including armA,rmtA,rmtB,rmtC and rmtD)and 9 kinds of AMEs gene[including aac(3)-Ⅰ,aac(3)-Ⅱ,aac(3)-Ⅲ,aac(3)-Ⅳ,aac(6')-Ⅰ b,aac(6')-Ⅱ,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ]were analyzed by PCR and verificated by DNA sequencing.Results In 40 strains of Enterobacter cloacae,the positive rates of genes of rmtB,aac(3)-Ⅱ,aac(6')-Ⅰ b,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ were 12.5%(5/40),27.5%(11/40),72.5%(29/40),32.5%(13/40),5.0%(2/40)and 5.0%(2/40),respectively.8 kinds of the rest of genes were all tested negative.The total positive rate of AMEs gene was 85.0%(34/40).Among 29 strains of Enterobacter cloacae that the aac(6')-Ⅰ b gene was positive,through PCR and verification by DNA sequencing,7 strains(24.1%)were confirmed to take the aac(6')-Ⅰ b-cr(the GenBank register number:EF375620,EU159121)alone,18 strains(62.1%)were confirmed to take the aac(6')-Ⅰ b-Suzhou(EU085533)alone,3 strains(10.3%)were confirmed to take both aac (6')-Ⅰ b-Suzhou and aac(6')-Ⅰ b-cr while only 1(3.4%)was aac(6')-Ⅰ b(the classical type).Conclusion There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world.At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-Ⅰ b-cr and aac(6')-Ⅰ b-Suzhou,with aac(6')-Ⅰ b-Suzhou gene was the predominance subtype in aac(6')-Ⅰ b.

Objective To investigate the status of genotype of the KPC(Klebsiella pneumoniae carbapenemase)-encoding genes in Pan-resistant K. Pneumoniae, isolated from the 98th Hospital of People' s Liberation Army, Huzhou district, Zhejiang province, China. Methods 19 strains of Pan-resistant K. Pneumoniae were isolated from the inpatients between November, 2008 and July,2009. Phenotypic confirmatory test for suspected carbapenemases production were carried out by Modified Hodge test. Carbapenemase gene of blaKPC was analyzed by PCR and verified by DNA sequencing. Results In 19 strains of K. Pneumoniae, the positive rates of Modified Hodge test and gene of blaKPC were both 100.0%. These genes all belonged to blaKPC-2 subtype confirmed by nucleotide sequence analysis. Among them, the blaKPC-2 gene sequence of the HZ001 strain (its original serial number was HZ9871 ) had been registered in GenBank (GenBank Accession Number: GU086225).Conclusion All of the Pan-resistant K. Pneumoniae isolated from the inpatients harbored blaKPC-2 type carbapenemases gene and causing an outbreak in a hospital. Carbapenemases that producing type KPC-2 might be the major reason which causing the resistance to Carbapenems antibiotics.

OBJECTIVETo examine modulations caused by cyclooxygenase-2 (COX-2) inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possible mechanisms.

METHODSCelecoxib (a COX-2 inhibitor) was administered 45 min prior to pilocarpine administration. The effects of COX-2 inhibitors on mIPSCs (miniature GABAergic inhibitory postsynaptic currents) of CA3 pyramidal cells in the hippocampus were recorded. Expressions of COX-2, c-Fos, newly generated neurons, and activated microgliosis were analyzed by immunohistochemistry, and expressions of alpha-subunit of gamma-amino butyric acid (GABA(A)) receptors and mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activity were detected by Western blotting.

RESULTSPretreatment with celecoxib showed protection against pilocarpine-induced seizures. Celecoxib prevented microglia activation in the hilus and inhibited the abnormal neurogenesis and astrogliosis in the hippocampus by inhibiting MAPK/ERK activity and c-Fos transcription. Celecoxib also up-regulated the expression of GABA(A) receptors. NS-398 (N-2-cyclohexyloxy-4-nitrophenyl-methanesulfonamide), another COX-2 inhibitor, enhanced the frequency and decay time of mIPSCs.

CONCLUSIONThe COX-2 inhibitor celecoxib decreased neuronal excitability and prevented epileptogenesis in pilocarpine-induced status epilepticus rats. Celecoxib regulates synaptic reorganization by inhibiting astrogliosis and ectopic neurogenesis by attenuating MAPK/ERK signal activity, mediated by a GABAergic mechanism.

Animals ; Blotting, Western ; Celecoxib ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Disease Models, Animal ; Fibrocystic Breast Disease ; metabolism ; Hippocampus ; drug effects ; enzymology ; pathology ; Immunohistochemistry ; MAP Kinase Signaling System ; drug effects ; Male ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Nitrobenzenes ; pharmacology ; Pilocarpine ; Proto-Oncogene Proteins c-fos ; metabolism ; Pyrazoles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; biosynthesis ; Status Epilepticus ; chemically induced ; enzymology ; pathology ; Sulfonamides ; pharmacology ; Synapses ; drug effects ; pathology

OBJECTIVETo investigate the prevalence of aminoglycoside resistance and genotyping of acetyltransferase in Escherichia coli.

METHODSResistance phenotypes to 12 antibiotics of 44 Escherichia coli isolates were analyzed using agar dilution method and 3 aminoglycoside resistance genes aac(3)-I, II and aac(6')-I were determined by PCR method.

RESULTSIn 44 clinical isolates, the occurrence of ESBLs was 45.45%, resistance rates were discrepant for amikacin (18.18%), gentamicin (56.82%) and tobramycin (61.36%), the prevalence of phenotype TG (tobramycin and gentamicin) indicative of aac(3)-II production and TGA (tobramycin, gentamicin and amikacin) indicative of aac(6')-I production were 36.36% and 18.18%, respectively. The most common aminoglycoside resistance genotype of acetyltransferase was aac(3)-II (52.27%) and aac(6')-I was lower (29.55%), but no aac(3)-I was detected.

CONCLUSIONAt least 2 acetyltransferase genes exist in this area i.e. aac(3)-II and aac(6')-I.

Acyltransferases ; genetics ; Amikacin ; pharmacology ; Aminoglycosides ; pharmacology ; Drug Resistance, Bacterial ; genetics ; Escherichia coli ; enzymology ; genetics ; Genotype ; Gentamicins ; pharmacology ; Phenotype ; Tobramycin ; pharmacology