P53 GENE MUTATIONS IN NON-SMALL CELL LUNG CANCER DETECTED BY POLYMERASE CHAIN REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non small cell lung cancer(NSCLC),74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5 8) using exon specific PCR, single strand conformation polymorphism (PCR SSCP). p53 mutations were observed in 55 4%(41/74) of the samples. No linkages were detected between the incidence of p53 mutations and histological type, lymph node metastasis,age or sex. Significant association between p53 mutations and degree of differentiation in adenocarcinomas, not in squamous cell carcinomas, was observed. The frequency of p53 mutations in smokers(65 3%) was higher than in nonsmokers(33 3%) and reached statistical significance.We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis.These results suggest that smoking could be an important factor in lung carcinogenesis,p53 mutation is a worse prognosis indicator in adenocarcinomas and related to high aggressive behavior of human lung cancer.

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

To isolate the cDNA partial sequence of key enzyme gene GalAT for pectin biosynthesis in ramie [Boehmeria nivea （L.） Gaud], and thus to understand the expression of GalAT gene in different tissues of ramie, degenerate primer was designed according to GalAT conserved sequence in other species reported, and the cDNA sequence of GalAT gene from ramie variety Zhongzhu 1 was cloned by RT-PCR method based on the degenerate primer. The cDNA revealed a 986-bp in length which encoded 328 amino acids. The cDNA sequence and putative amino acid sequence of GalAT shared high identity with previously reported Arabidopsis thaliana GA UT4 （GalAT） as 77 and 83%, respectively. Molecular evolution analysis showed that the putative amino acid sequence and Arabidopsis thaliana GAUT4 gathered to a same group. Real-time quantitative PCR analysis showed that GalAT mRNA accumulated most abundantly in root, and GalAT transcripts in all kinds of ramie tissues in turn revealed as follows： root 〉 leaf〉 bast 〉 or ≈ xylem.

Identification,Characterization, and Expression of P450 Gene Encoding CYP6BQ13v2 from the Red Flour Beetle,Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae)

An allele of CYP6BQI3, named CYP6BQ 13v2 （GenBank accession no. FJ209361）, was isolated from the red flour beetle, Tribolium castaneum （Herbst） （Coleoptera： Tenebrionidae） by RT-PCR. The cDNA sequence of CYP6BQ13v2, 1 563 bp in length, contains an open reading frame of 1 554 nucleotides encoding a putative protein of 518 amino acid residues with a predicted molecular weight of 59.92 kDa and a theoretical pl of 7.60. The putative protein contains the classic hemebinding sequence motif F××G×××C×G （residues 456-465） conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 98% identity with the previously published sequence of CYP6BQ13 （GenBank accession no. XP967146） from the T. castaneum genome project. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that there was closer phylogenetic relationship of CYP6BQ 13v2 with CYP302A1 and CYP307A1 mediating synthesis of the insect molting hormone, distant relationship with CYP6B1 metabolizing plant allelochemicals, CYP6D 1 linking to pyrethroid resistance and other members of CYP6 family. Expression test of the gene in the adults and immature stages of T. castaneum by quantitative real-time PCR revealed that CYP6BQ13v2 is expressed in all life stages investigated. The mRNA expression level in 1st instar larvae was 14.9- and 3.86-fold higher than those in pupae and adults, respectively. The CYP6BQ13v2 expression levels appeared in the order of 1st instar larvae, followed by 4th instar larvae, 7th instar larvae, adult, and pupae from high to low. The more bioinformation of CYP6BQ 13v2 was also analyzed.

Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases

AIM： To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Flelicobacter pylori （H pylori） 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography （ERCP） from 60 Indian subjects. METHODS： Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori （16S rRNA, urea and cagA） genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepatobiliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups Ⅰ and Ⅱ respectively. Ten from group Ⅰ were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori. CONCLUSION： Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.

Development of a PCR-based method to monitor arthropod dispersal in agroecosystems:Macrolophus pygmaeus(Hemiptera:Miridae)from banker plants to tomato crops

Development of conservation biological control programs requires the identifi-cation of sources that contribute to predator colonization of crops.Macrolophus pygmaeus(Rambur)(Hemiptera:Miridae)is an efficient polyphagous predator used in biological con-trol programs in vegetable crops in Europe.We have developed a marking method based on spraying with a solution of the brine shrimp Artemia spp.(Anostraca:Artemidac)cysts,followed by a PCR detection of Artemia DNA to monitor M.pygmaeus dispersal from banker plants to tomato crops.Experiments conducted in climatic chambers show that the topical application of this marking solution on M.pygmaeus does not significantly reduce adult longevity and that it is detected up to 6 d after the application.When this Artemia solution was applied on Calendula officinalis L.banker plants harboring M.pygmaeus and maintained outdoors,Artemia DNA was still detected on 62%of the insects after6 d.The conducted field applications in commercial greenhouses have confirmed the usefulness of this method to monitor M.pygmaeus dispersal from banker plants to a newly planted tomato crop.This method can be used to assess arthropod movement,being an interesting molecular approach for further improving future pest management strategies.