To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR (polymerase chain reaction). The suspension of a single colony in LB medium was used as the template, SP6 (the upstream primer of the plasmid cDNA library) and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank (accession number: JX197456), and it was indicated as a partial cDNA sequence with a deletion at the 5' end. The transcript is 997 bp consisting of 31 bp 5', 618 bp 3' untranslated region (UTR) and an open reading frame (ORF) of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three a-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus (Silurana) tropicalis and 89% with Xenopus laevis, and 71%-85% with other species.

Objective To investigate the protective effect of flos puerariae flavonoid on adriamycin (ADR)-induced toxic myocarditis and its mechanisms from morphological,biochemical and molecular levels.Methods 96 healthy Kunming male mice were randomly divided into 6 groups:normal control group,ADR model control group,ADR+low dose of flos puerariae flavonoid group(50 mg/kg),ADR +middle dose of flos puerariae flavonoid group(100 mg/kg),ADR +high dose of flos puerariae flavonoid group(200 mg/kg),and Vit E positive control group(40 mg/kg),16 in each group.The drugs were orally administered for consecutive 15 d and the model of toxic myocarditis was induced by intraperitoneal injection of ADR(3 mg/kg)in mice from day 2,one time every other day,for 7 times Colorimetry was used to measure the changes of marker enzymes about myocardial injury and inducible nitric oxide synthase(iNOS)activity in serum and tissue;immunohistochemical method was adopted to detecte the expression of myocardial apoptosis related proteins Bax and bcl-2;HE staining was conducted to observe the pathological changes of cardiac structure.Results Compared with normal control group,ADR(3 mg/kg,ip,7 times)induced the elevation of serum creatine kinase (CK),lactate dehydrogenase (LDH),aspartate transaminase(GOT)and iNOS activity increased significantly in mice(P<0.01).Meanwhile myocardial superoxide dismutase(SOD)activity decreased, and the malondialdehyde(MDA)content increased(P<0.01).Myocardial cell apoptosis in mice increased significantly,and the apoptosis rate was(40.5 ± 5.2)%;the expressions of Bax and Bcl-2 were significantly increased(P<0.01),However,the Bcl-2/Bax ratio decreased.The flos puerariae flavonoid (50,100,200 mg/kg,ig,15 d)and Vit E positive control group could reverse the changes induced by ADR,decrease serum CK,LDH,GOT and iNOS activities,increased myocardial SOD activity,lower MDA content and the expression of bax protein,and elevated Bcl-2/Bax ratio,in a dose-dependent manner.Light microscopy confirmed that flos puerariae flavonoid significantly alleviated the changes of myocardial microstructure.Conclusion ADR could induce myocardial cell apoptosis and lead toxic myocarditis in experimental mice.The flos puerariae flavonoid has protective effect on ADR-induced myocardial injury and the mechanism may be related to elevating myocardial SOD activity and anti-lipid peroxidation,inhibiting the expression of Bax protein and adriamycin-induced cardiomyocyte apoptosis.

Objective To understand the genetic background of the specific pathogen-free Yorkshire and Landrace pigs, imported from Canada by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.Methods The population genetics were investigated by using 19 pairs of microsatellite primers.Results In the Yorkshire pigs and Landrace pigs, 84 and 89 alleles, respectively, were detected at 19 microsatellites loci.The average polymorphic informa-tion content and mean heterozygosity in the Yorkshire pigs were 0.5271 and 0.5877, and in the Landrace population were 0.5652 and 0.6066, respectively.Because of the significant ( P<0.01) differences of alleles in different loci such as S0155,S0143,S0178,Sw857 and Sw936, it made them possible to be used to identify Yorkshire and Landrace pigs′breed. F-statistics showed that the differentiation within the population was small and genetic structure was stable.Conclusions Compare with the domestic pedigree large White and Landrace pigs, the SPF pedigree pigs imported from Canada are more stable in genetic structure, and can be used as laboratory animal models in animal science research.

Objective To investigate the prognostic factors and the clinical outcome of locally recurrent rectal cancer after radical resection. Methods From April 2000 to April 2004, 105 patients with locally recurrent rectal cancer after radical resection were re-treated in Tianjin cancer hospital. Thirty-four patients were re-treated with surgery combined with adjuvant chemoradiotherapy (group 1), 35 with surgery alone (group 2), and 36 with chemoradiotherapy (group 3). The impact of 17 clinicopathological factors and treatment modalities on the survival was analyzed. Results The follow-up rate was 95. 2%. The median survival time was 23 months. The 1-, 3-and 5-year survival rates of patients with locally recurrent rectal cancer were 63% ,34% and 19%, respectively. The 1-, 3-and 5-year survival rates were 79%, 55% and 32% in group 1 ; 68%, 40% and 14% in group 2; and 64%, 36% and 11% in group 3; respectively (χ~2 =7. 96,P =0. 019). The univariate analysis showed that the degree of differentiation, depth of tumor invasion, number of metastatic lymph nodes, initial TNM stage, recurrent location, time to recurrence, and surgery combined with adjuvant therapy were significant prognostic factors, with the last 4 being the independent prognostic factors. Conclusions Surgery combined with chemoradiotherapy may improve the survival of patients with locally recurrent rectal cancer.

Objective To evaluate the role of large conductance calcium-activated potassium (BKCa) channels in vascular hyporesponsiveness in rats with obstructive jaundice.Methods Eighteen male Sprague-Dawley rats,weighing 180-200 g,were randomly divided into 3 groups (n =6 each) using a random number table:control group (group C),sham operation group (group S),and bile duct ligation group (group BDL).Obstructive jaundice was produced by common bile duct ligation.At 7 days after surgery,blood samples were collected for determination of the levels of serum total bilirubin (TBL),direct bilirubin (DBL),indirect bilirubin (IBL),alanine aminotransferase (ALT),and aspartate aminotransferase (AST).Thoracic aortic rings were prepared,and the endothelium was removed.The aortic rings were sequentially perfused with different concentrations of norepinephrine (NE) and sodium nitroprusside (SNP),and the maximum amplitude of contraction and dilatation of aortic rings was recorded.The aortic rings were then perfused with BKCa channel blocker Chtx with the final concentration of 10 7 mol/L,followed by perfusion with different concentrations of NE and SNP again,and the maximum amplitude of contraction and dilatation of aortic rings was recorded under each concentration.The percentage of maximum contraction and dilatation (maximum amplitude after Chtx administration÷maximum amplitude before Chtx administration× 100％) was calculated.Results Compared with C and S groups,the levels of TBL,DBL,IBL,ALT and AST in serum were significantly increased,the maximum amplitude of NE-induced contraction of aortic rings was decreased,and the percentage of the maximum NE-induced dilatation of aortic rings was increased,the maximum amplitude of SNP-induced contraction of aortic rings was increased,and the percentage of the maximum SNP-induced dilatation of aortic rings was decreased in group BDL.Conclusion Excessivc opening of BKCa channels may be involved in the mechanism of vascular hyporesponsiveness in rats with obstructive jaundice.