Objective To investigate the relationship between truncating mutations of APC gene and sporadic colorectal cancer,and analyze the feasibility of non-radioactive protein truncation test (PTT) in the detection of mutations of APC gene.Methods Ninety-six samples of sporadic colorectal cancer tissues ( including 44 patients with colonic cancer and 52 patients with rectal cancer) were obtained from the General Hospital of Ningxia Medical University from September 2008 to September 2010.The mutation cluster region of the APC gene was screened using digital PTT labeled with fluorescent Lys-t-RNA,with a polymerase chain reaction fragment amplified from genomic DNA serving as a tenplate for in vitro translation.The occurrence of gene mutation was determined according to the emergence of truncated peptides.The mutation cluster region of the APC gene in 46 samples of colorectal cancer tissues was analyzed by direct sequencing.The detection rates of the 2 methods were compared by chi-square test.Results Thirteen (26％) truncated peptides were detected in the 50 samples of colorectal cancer tissues.The mutation type of 4 samples is nonsense mutation,which resulted in emergence of truncated gene products.Eleven (24％) truncated peptides were detected in the 46 samples of colorectal cancer tissues.There was no significant difference in the detection rates between PTT and direct sequencing ( x2 =0.033,P ＞ 0.05 ).Conclusions Truncating mutations of APC gene are common alterations in sporadic colorectal cancer in the Chinese.Digital PTT labeled with fluorescent Lys-t-RNA is rapid and high-sensitive in screening gene mutations.

BackgroundIt has been well-known that integrin linked kinase(ILK) plays an important role in the pathogenesis of neorascularization.But there are few researches to elucidate the relationship between ILK and retinal nevascularization. Objective This study was to explore the expression and significance of ILK on retinal neovascularization and new vessels regression in the oxygen-induced retinopathy(OIR) mouse model. Methods One hundred and twenty-eight 7-day-old C57BL/6J mice were randomly divided into the normal group and model group.The mice were rose in(75±2)％O2 environment with mother mice together for 5 days and then in normal environment for 5 days to establish the OIR models.The mice in normal group were rose in the normal environment for 21 days.The 17-day-old mice were sacrificed and retinal sections were prepared for histopathological examination and the numbers of the cellular nuclei of vascular endotheliumbreaking retinalinternal limiting membranewere calculated.Retinal sections and sheeting were prepared in 12,14,17 and 21 -day-old mice to examine the formation and regression of new blood vessel using ADP histochemistry staining,and then immunochemistry,real-time PCR and Western-blotting were used to detect the expressions of ILK and its mRNA in retina. ResultsThe numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were (45.64 ± 12.17 )in OIR group,and those of the normal group were( 0.35±0.14 )with a significant difference between two groups (t =22.85,P＜0.05).Retinal new blood vessel appeared in 14-day mice,and peaked in 17-day mice and then regression in 21-day mice.ILK protein was expressed mainly in retinal ganglion cell layer,inner nuclear layer,inner plexiform layer and photoreceptor layer.Real-time PCR showed that ILK mRNA expressing levels in retina in model group were( 1.00±0.22),(1.85±0.17),(1.58±0.43) and(1.53±0.36) respectively in 12-,14-,17- and 21-day mice.Westernblotting determined that the A value of the ILK/β-actin was increased in 12-,14-,17-day mice and decreased in 21-day mice,and the A values were significantly higher in model group than the control group in various aged mice ( t =2.97,P＜0.05 ;t =11.88,P＜0.01 ;t =16.84,P＜0.01 ;t =13.00,P＜0.01 ). ConclusionsThese results indicate a space-time corresponding relation between the expression of ILK and retinal neovascularization.The obvious positive expression of ILK may be highly correlated with retinal neovascularization.

To obtain a number of extracellular penicillinase,the gene(penp)encoding penicillinse of Bacillus cereus ATCC10987 was cloned into expression vector in Bacillus subtilis,transformed into Bacillus subtilis DB104 deficient in two proteases.When recombinant bacteria was cultured in LB medium for 24 hours,the result of SDS-PAGE showed that the molecular weight of the protein was 28kDa and the penicillinase activity reached 339U/ml.By screening seven different fermentation media,the result showed that 4# medium is favorable to producing penicillinase by the recombinant cells than the others,with the yield of the enzyme being 1580U/ml.When the recombinant cells was cultured in 7 liter fermentor for 24h,the penicillinase activity reached 1255.8U/ml.

The NSP2 gene of porcine reproductive and respiratory syndrome virus (PRRSV)S1 strain was partly amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli. The purified GST-tNSP2 protein showed a strong reaction with the PRRSV-positive sera in Western blot assay. Balb/c mice were immunized with the purified protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5. They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA, but not with the PRRSV SY0608 strain. Both of the McAbs belong to IgG1 isotype, and their light chains belong ? type. The expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.

Objective To identify and rename one strain stored in National Center for Medicine Culture Collections ( CMCC) and used for tracheitis vaccine production. Methods The test strain CMCC (B)29108 and the type strain DSM30007T were cultured on NA medium. Characteristics in morphology, physiology, biochemistry and fatty acid profile were compared between the two strains. Phylogenetic analysis was based on 16S rRNA and rpoB gene sequences, together with the DNA-DNA hybridization assay. Results A Comparative analysis of a partial sequence of the 16S rRNA gene sequence revealed that the CMCC( B) 29108 strain was closed to the Acinetobacter species and showed the highest similarity with the type strain Acinetobacter baumannii DSM30007T. Moreover, the CMCC(B)29108 strain was highly similar to type strain DSM30007T in morphology, physiology, biochemistry and fatty acid profile. On the phylogenetic tree based on 16S rRNA and rpoB gene of all Acinetobacter members, the CMCC(B)29108 strain steadily clustered into one independent branch only with the DSM30007 T strain with a DNA-DNA hybridization value of 100%. Conclusion The CMCC(B)29108 strain that is one of the strains used for the production of tracheitis vac-cine should be assigned to the species of Acinetobacter baumannii based on its phenotypic, phylogenetic and chemotaxonomic characteristics.

Objective To characterize the antibiotic resistance,homology and carbapenemase genotypes of imipenem resistant Acinetobac1ter baumannii isolated from our hospital,and analyze the clonal relatedness of the test strains.Methods Ninety five strains of imipenem resistant A.baumannii were isolated from August 2003 to December 2004 in the First Affiliated Hospital, College of Medicine,Zhejiang University.The MICs of 16 antimicrobial agents against these strains were determined by agar dilution and E-test method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding gene of carbapenemases was amplified.PCR products were purified,cloned and sequenced.Plasmid DNA was extracted and purified.Conjugation and Southern blot were performed to locate the position of oxa 23 gene.Results The resistance rates to ampicillin-sulbactam and cefoperazone sulhactam were 67.9% and 30.2%.Polymyxin E had the lowest resistance rate of 17%. The resistance rate to other antimicrobial agents was higher than 90%.The 95 strains,isolated from 10 clinical units,were classified into 6 clones.Clones A and B were predominant clones.All strains produced carbapenemases which were confirmed as OXA 23 by PCR and sequencing analysis.No plasmid was extracted and conjugation was not successful.Southern bolt showed that oxa-23 gene was located on Apal-digested chromosomal segments about 220 kb and 200 kb in Clones A and B,re spectively.Conclusions OXA 23-producing A.baumannii has become one of the most important multi-resistant pathogens in our hospital.Clones A and B have widely spread in our hospital.Oxa-23 gene is located on chromosomal DNA.

Objective To investigate the resistant mechanism of imipenem-resistant K. pneumoniae.Methods The minimal inhibitive concentrations (MICs) of the antimicrobial agents were determined by Etest.Isoelectric focusing electrophoresis (IEF),plasmid extraction,conjugation, transformation,PCR amplification,cloning and sequencing were carried out for analyzing the encoding gene of ?-1actamases.Results Three kinds of ?-1actamases were detected with pIs of 7.2,6.7,and 5.4.in a clinical strain of K.pneumoniae.These ?-1actamases were TEM-I (pI,5.4),SHV-12 (pI,8.2) and KPC-2 ( pI,6.7 ) confirmed by sequencing of the PCR products.Only one band of ?-1actamase with pI 6.7 was displayed in the transformant.A 1500 bp segment,which contained the KPC-2 gene confirmed by nucleotide sequence analysis,was cloned from a 60 000 bp plasmid of the transformant.Conclusion The strain of K.pneumoniae resistant to imipenem produces a plasmid-mediated carbapenemase KPC-2 which belongs to Bush group 2f,class A ?-1actamase.

Objective To evaluate the risk of plague occurrence via surveying and analyzing indoor rat density and flea index in natural villages having previous plague experience. Methods During August to September 2007, 30 natural villages experiencing previous plague were selected based on the surveillance data, and then all households were coded with numbers and 20 households in each village were randomly selected via computer. Cages and sticky papers were set in 600 selected households to capture rats and fleas. Rat density, flea prevalence, flea index and median were estimated. Results One hundred thirty-three Rattus flavipectus and 33 Suncus murinus were caught and averaged rat density was 2.8 rats per one hundred cage. nights (166/6000), the median was 5 rats each village. One hundred and one mice infected fleas, flea prevalence on rats was 60.8% (101/166), 296 Xenopsylla cheopis and 48 Leptopsylla segnis were collected. Rat flea index was 2.1 fleas per rat (344/166). A total of 315 dissociated flea was caught, average dissociated flea index was 0.026 fleas per sticky paper (315/11888). The median was 5.5 dissociated fleas per village. Of dissociated fleas, Ctenocephalides felis felis (205) and Xenopsylla cheopis (103) accounted for 97.8% (308/315). The proportion for species of the rat flea and the dissociated flea was different(Fisher test: P < 0.01). The rat flea was significantly associated with the rat density(r = 0.68, P < 0.01), but the dissociated flea was significantly associated with neither the rat density(r = -yield than fried wheat batter(χ2 = 5.59, P < 0.05). Conclusions In these villages having previous plague experience of Lianghe County, Rattusflavipectus was dominant species of indoor rats, Xenopsylla cheopis and Ctenocephalides felis felis were dominant species of rat flea and dissociated flea, respectively. Mengsong, Bangdu, and Tangjiatun village had potential risk of plague emergence.

Aged ; CDX2 Transcription Factor ; Cysts ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Female ; Homeodomain Proteins ; metabolism ; Humans ; Keratin-20 ; metabolism ; Mucous Membrane ; pathology ; Peritoneal Neoplasms ; metabolism ; pathology ; surgery ; Pseudomyxoma Peritonei ; metabolism ; pathology ; surgery ; Rupture ; Splenic Neoplasms ; metabolism ; pathology ; surgery

Objective To compare the infectivity between laboratory adapted human inununodefi- ciency virus(HIV-1) and primary HIV-1 isolates for different mucosal epithelial cell lines. Methods Mu-cosal epithelial cells Caco-2, T-84, HeLa and lymphocyte MT-4 were infected with laboratory adapted HIV-1 SF33 and 2 primary HIV-1 isolates (02010561, 02010141). Culture supernatant and cells were collected respectively on 3-4 days interval after virus inoculation. The former was tested for HIV-1 antigen P24 level and viral load, and the latter was tested for total viral DNA and integrated viral DNA. Results All 3 virus strains could infect MT-4 cells and integrate into their genome. Only HIV-1 SF33 could infect Caco-2 cells but could not integrate into their genomic DNA. Both HIV-1 SF33 and 02010561 infected HeLa cells but only integration of HIV-1 SF33 was detected. All the 3 HIV-1 strains infected T-84 cells but only the integra-tion of HIV-1 SF33 and 02010141 was observed. Conclusion Although laboratory adapted and primary HIV-1 strains are able to infect human mucosal epithelial cell lines, transient or productive infection estab-lished in different mucosal epithelial cells is dependent on the character of cells and virus strains.