ObjectiveTo observe the preventative and therapeutical effect on bile pigment calculus of Jinqianduizuo Mixture which is a traditional Chinese medicine empirical formula and provide foundation for the therapy of cholelithiasis. MethodUsing the model of bile pigment calculus in guinea pigs. Observing the gall bladder’s shape,the rate of forming gallstone and the total bile. Then making biochemical determination of the bile and the blood serum. Result There are significant differences between model control group and three treated groups of Jinqianduizuo Mixture in the rate of forming gallstone,the bilious quantity,the contents of total bilirubin and total bile acids. While in the contents of direct bilirubin,total cholesterol,triglycerides and high-density lipoproteins,they have no significant difference. ConclusionThe results indicate that Jinqianduizuo Mixture can conspicuously cut down the gallstone’s incidence of guinea pigs and can obviously promote the bilious externalization in guinea pigs. It also can promote bile secretion distinctly,but have no evident effect on the ingredients of blood-lipid.

[Objective] To elucidate the effects and mechanism of Naoqingling Decoction(ND)on Gilles de la Tourette syndrome(TS).[Method] Central nervous system of mice was stimulated by amphetamine(AMP)to develop the model of experimental tourette syndrome(ETS).Then we observed the stereotypia and autonomic activities of mice,and determined the levels of monoamine neurotransmitters such as dopamine(DA),norepinephrine(NE),5-hydroxytryptamine(5-HT)and 5-hydroxyindoleacetic acid(5-HIAA)in the brains.[Result] To compare with model group,three-dose ND groups can resist the determinate activities stimulated by AMP;can obviously decrease the autonomic activities of mice and degrade the levels of DA,NE,5-HT and 5-HIAA in the corpora striates.[Conclusion] ND has therapeutical effect on ETS model and it's mechanism may be its effects on the secretion or metabolism of neurotransmitter in the brains.

[Objective] To discuss mechanism of FS-1272 eye drops on cataract of rats induced by D-galactose.[Method]By using the model of cataract of rats induced by D-galactose to determine the content of SOD,CAT,GSH-px,MDA in lens.[Result]FS-1272 eye drops can increase the activities of SOD,CAT,GSH-px and decrease the content of MDA.[Conclusion]FS-1272 eye drops have the effect of delaying the generation and development of cataract by alleviating the extent of oxidative injury of lens.

Objective: To describe the prevalence of permanent tooth caries and associated factors among junior high school students in Haikou, and to provide reliable evidence for prevention and control of caries. Methods: A total of 3 573 students from 8 junior high school in Haikou City and towns were selected by the methods of clustered sampling survey. Questionnaire survey and oral health examinations were conducted to analyze the caries rate, mean decayed missing filled tooth (DMFT), filling rate, pit and fissure sealant rate. Logistic regression was used to analyze the influencing factors of caries in permanent tooth. Results: Among the surveyed junior high school students, the caries rate was 47.32%( n =1 691), the mean DMFT was 1.51. The caries rate and mean DMFT were higher in female students (49.59%) than in male students (44.95%), and higher in towns (50.77%) than in urban areas (44.04%), the difference were statistically significant ( χ 2=16.25, 7.72, P <0.05). The filling rate and pit and fissure sealant rate among junior high school students in Haikou were 17.13%, 6.27% respectively. The filling rate and pit and fissure sealant rate were higher in urban areas (18.97%, 7.17%) than towns (15.76%, 5.33%), the difference were statistically significant ( χ 2= 9.57, 5.13, P <0.05). The multivariate Logistic analysis showed that female student, town residence, daily consumption of sweets or sugary drinks (≥1 time), bedtime snack were risk factors for junior high school students suffering from permanent dental caries( OR =1.41, 1.45, 2.63, 2.09, 1.53), while using fluoride toothpaste daily, oral education in school were protective factors( OR =0.44, 0.34)( P <0.05). Conclusion The caries rate of permanent teeth among junior high school students in Haikou is at high level, but the filling rate of caries and pit and fissure sealant rate are lower. The prevention and treatment of dental caries should be carried out in high risk students, and oral health education in school is also needed to improve the oral health level of junior high school students.

BACKGROUNDTo summarize the clinical results of bronchoplastic procedures and pulmonary artery reconstruction or combined with other resection and plasty of heart, great vessels in the treatment of 304 patients with locally advanced lung cancer.

METHODSFrom February, 1983 to December, 2001, double sleeve resection and reconstruction of bronchus and pulmonary artery, or combined with other resection of heart, great vessels were carried out in 304 patients with locally advanced lung cancer. The operations included double sleeve left upper lobectomy in 199 cases; double sleeve right upper lobectomy in 21 cases; double sleeve right upper middle lobectomy in 14 cases; double sleeve left upper lobectomy combined with resection of left atrium in 8 cases; double sleeve right upper lobectomy combined with superior vena cava (SVC) resection and reconstruction with Gortex graft in 29 cases; double sleeve right upper middle lobectomy combined with SVC resection and reconstruction in 21 cases; double sleeve right upper middle lobectomy, carinal and SVC resection and reconstruction in 11 cases; left pneumonectomy combined right main pulmonary artery and pulmonary artery trunk resection and reconstruction with Gortex graft in 1 case.

RESULTSThere were 3 operative deaths. The operative mortality was 1% in this series. Sixty four patients had operative complications. The operative complication rate was 21.05% (64/304). The 1-, 3-, 5- and 10 year survival rates were 81.75%, 60.14%, 37.21% and 24.39% respectively.

CONCLUSIONSDouble sleeve lobectomy or comblined with other resection and reconstruction of heart, great vessels can significantly improve the prognosis and increase the curative rate and long term survival in patients with locally advanced lung cancer.

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Base Sequence ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDScreening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study.

METHODSSubtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments.

RESULTSThe subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method.

CONCLUSIONSSSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.

BACKGROUNDRas to MAPK pathway plays a critical role in the transmission of many growth and developmental signals. A new component of this pathway which is termed kinase suppressor of Ras (KSR) was found in 1995. KSR is as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase (MAPK) and its upstream regulators. It has been proven that KSR has many phosphorylation sites, and phosphorylation state changes response to signaling events. Site-directed mutagenesis can precisely change the base sequence and get mutant proteins. The aim of this study is to construct two mutant proteins of KSR by using site-directed mutagenesis, and to express and purify them, therefore to provide basement for studying the functional and biochemical mechanisms of KSR.

METHODSSite-directed mutagenesis of pCMV-Tag2b-KSR gene was performed by modified QuikChangtm site-directed mutagenesis kit method. Two pairs of mutagenic primers were synthesized in vitro and two mutations desired, the recombinant plasmids were verified by restriction enzyme analysis and DNA sequencing. Then positive clones were transfected into 293T cell line. The purified mutant proteins were analyzed by Western blot.

RESULTSTwo mutants were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant genes were completely concordant with experiment design, which could be used to be transfected into 293T cell line. The purified mutants were identified by Western blot.

CONCLUSIONSTwo mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.

BACKGROUNDThe present experimental data have showed that the function of kinase suppressor of Ras (KSR) is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line.

METHODSN-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot.

RESULTSThe sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot.

CONCLUSIONSEukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.

BACKGROUNDPrevious researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.

METHODSSite-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.

RESULTSFive nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.

CONCLUSIONSFive nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.