Sequence Analysis and Quantitative Detection of Norwalk-like Viruses in Cultured Oysters of China

We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） region of NLVs genomes with RT-PCR, the open reading frame 1 （ORF 1） of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts （copies） of NLVs in positive patient＇s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.

Use of arrays to investigate the contribution of ATP-binding cassette transporters to drug resistance in cancer chemotherapy and prediction of chemosensitivity

Multidrug resistance （MDR） is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette （ABC） transporters. While some members of human ABC transporters have been shown to cause drug resistance with elevated expression, it is not yet known whether the over-expression of other members could also contribute to drug resistance in many model cancer cell lines and clinics. The recent development ofmicroarrays and quantitative PCR arrays for expression profiling analysis of ABC transporters has helped address these issues. In this article, various arrays with limited or full list of ABC transporter genes and their use in identifying ABC transporter genes in drug resistance and chemo-sensitivity prediction will be reviewed.

Two distinct pathways of p16 gene inactivation in gallbladder cancer

AIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer,and to investigate p16 alterations and their correlation with clinicopathological features. METHODS: Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression,loss of heterozygosity (LOH),homozygous deletion and promoter hypermethylation using immunohistochemistry,microsatellite analysis,quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively. In addition,mutations were examined by direct DNA sequencing. RESULTS: Homozygous deletions of the p16 gene exon2,LOH at 9p21-22,p16 promoter hypermethylation,and loss of p16 protein expression were detected in 26.0% (13/50),56.9% (29/51),72.5% (37/51) and 62.7% (32/51),respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene,a combination LOH and promoter hypermethylation,and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene,respectively. P16 alterations were not associated with clinicopathological features. CONCLUSION: Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion,a combination of LOH and promoter hypermethylation,and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.

Methylation of RAR-β2, RASSF1A, and CDKN2A Genes Induced by Nickel Subsulfide and Nickel-carcinogenesis in Rats

Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide （Ni 3 S 2 ） at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5’ region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of the three genes both in muscle and lung tumor were decreased distinctly in comparison with normal tissue. But hypermethylation was found only in muscle tumor. Conclusion These findings suggest that loss of function or decrease of RAR‐β2, RASSF1A, and CDKN2A, as well as the hypermethylation of 5’ region of these genes, are related with nickel exposure.

Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma （CCA） and define its correlation with clinical parameters. METHODS： Quantitative real time PCR （qPCR） was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the X^2 test. Cox regression analysis was used for determining patient＇s survival. RESULTS： Amplifications of the TP （22q13.33）, TOB2 （22q13.2-13.31）, D22S283 （22q12.3）, TIMP3 （22q12.3） and NF2 （22q12.2） were found in 35 （53.8%）, 28 （43.1%）, 27 （41.5%）, 24 （36.9%）, and 24 （36.9%）, respectively. Losses at the D22S423 （22q13.1-13.2）and BIK （22q13.31） were detected in 26 （40%） and 23 （35.4%）, respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK （P = 0.025） and D22S283 （P = 0.041）. Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis （P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779） and blood vessel invasion as an independent poor prognostic factor （P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571） in CCA patients. CONCLUSION： This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA, This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.

Molecular Cloning and Expression of Squalene Epoxidase from a Medicinal Plant, Bupleurum chinense

Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.