[Characterization of reaction kinetics between ligand and receptor on the solid and liquid interface based on optical waveguide lightmode spectroscopy].

The present paper describes the use of optical waveguide lightmode spectroscopy (OWLS) for study of the binding interaction of the vascular endothelial growth factor (VGEF) with VEGF receptor 2 (VEGFR2). VEGF were immobilized in the surface of an 3-amino 3-propyltriethoxy silane (APTES) modified sensor chip. The solutions with different concentration of VEGFR2 were injected to the system to investigate the kinetic character with OWLS on the solid and liquid interface. The receptor binding and dissociation on the interface, quantified by association and dissociation rate coefficients ka and kd, were determined by the OWLS experiments. The k(a) and k(d) is 6.86 x 10(5) L x mol(-1) x s(-1) and 1.15 x 10(-3) s(-1), respectively. The results show that OWLS method could meet the requirement of kinetic determination of ligand--receptor interaction in applications for related fundamental research and pharmaceutical development.

[Electrochemical detection of toxin gene in Listeria monocytogenes].

Listeria monocytogenes (LM) is a food-borne pathogen inducing listeriosis, an illness characterized by encephalitis, septicaemia, and meningitis. Listeriolysin O (LLO) is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. One of the best ways to detect and confirm the pathogen is detection of one of the virulence factors, LLO, produced by the microorganism. This paper focused on the electrical method used to detect the LLO toxin gene in food products and organism without labeling the target DNA. The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto the gold electrode with the mercaptan activated by N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N'-ethyl carbodiimidehydrochloride (EDC). The hy-bridization reaction that occurred on the electrode surface was evidenced by Cyclic Voltammetry (CV) analysis using [Co(phen)3](ClO4)3 as an indicator. The covalently immobilized single-stranded DNA could selectively hybridize to its complementary DNA in solution to form double-stranded DNA on the gold surface. A significant increase of the peak cur-rent of Cyclic Voltammetry (CV) upon hybridization of immobilized ssDNA with PCR amplification products in the solu-tion was observed. This peak current change was used to monitor the amount of PCR amplification products. Factors deter-mining the sensitivity of the electrochemical assay, such as DNA target concentration and hybridization conditions, were investigated. The coupling of DNA to the electrochemical sensors has the potential of the quantitative evaluation of gene.

Isolation and functional analysis of sulfite oxidase gene AtSO promoter from Arabidopsis thaliana.

Sulfite oxidase (SO), one of the known molybdenum co-factor-containing enzymes, plays important roles in diverse metabolic processes such as sulfur detoxification and purine catabolism in mammals. But much less is known about the expression and regulatory characterization of sulfite oxidase gene in higher plants. In this report, expression of Arabidopsis SO is characterized in detail by semi-quantitative RT-PCR and histochemical staining. The results showed that the transcripts of AtSO were predominantly detected in Arabidopsis aerial tissues including stems, young leaves, young inflorescences and immature siliques at higher level, but in roots with a lower level. To monitor AtSO expression in plant, the promoter region containing a 1 562-bp genomic sequence from AtSO was isolated and analyzed using methods of bioinformatics. Basing on the distribution of beta-glucuronidase (GUS) activities shown by histochemical staining in transgenic Arabidopsis plants harboring the promoter-uidA fusion construct, it can be concluded that AtSO is expressed mainly in the green tissues/organs in a light-dependent way. In addition, its expression is up-regulated during sulfite treatment. The information from this study may provide useful clue for further functional analysis of plant SO homologs during light-induced development of leaf tissue and/or excessive sulfite/SO(2) gas stresses in higher plants.

Rapid desalting and protein recovery with phenol after ammonium sulfate fractionation.

Ammonium sulfate (AS) fractionation is often used in protein and enzyme purification; however, the resultant protein pellets have a high salt content and desalting by dialysis is required prior to next analysis. Here, we have developed a phenol-based method for quick desalting and concentrating proteins after AS fractionation of complex olive leaf protein extract. After redissolving, AS precipitates were desalted with phenol extraction and the abundance of beta-glucosidase in each fraction was monitored with a specific antibody. The results of electrophoresis and Western blot showed the feasibility of the method. The method is quick and universal, and does not need much technique.

[Expression and subcellular localization of the ORF4 gene of barley yellow dwarf virus GAV strain in baculovirus-insect cell expression system].

According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four a-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.

Hepatitis gene chip in detecting HBV DNA, HCV RNA in serum and liver tissue samples of hepatitis patients.

OBJECTIVE: To study the preparation of diagnostic gene chip for detecting hepatitis B virus (HBV) and hepatitis C virus (HCV) and its accuracy in detecting HBV DNA and HCV RNA in serum and liver tissues. METHODS: The probes, which depend on the conservative gene fragment of hepatitis virus, was designed, synthesized and spotted on the modified glass. The probes and some other control probes were assembled on the diagnostic microarray of hepatitis virus. The gene of hepatitis virus, purified from blood or tissue, was labeled with fluorescence and hybridized to the microarray. The hybridized microarry was scanned with microarray scanner and the diagnostic result was analyzed from the scanning data. Fourty patients with hepatitis B virus and 40 healthy people or 40 patients with hepatitis C virus were subjected to detection of HBV DNA and HCV RNA with the hepatitis virus gene chip by the double-blind method. Paraffin liver specimens obtained from 99 cases of posthepatitic cirrhosis were used to detect HBV DNA. The liver tissues and serum from 15 cases of chronic hepatitis B were used to detect HBV DNA. Simultaneously, HBsAg and HBcAg were detected in the serum by fluorescence microparticle quantitation, HBV DNA and HCV RNA in the serum by PCR, and HBcAg in liver tissues by immunocytochemistry or HBV DNA by in situ molecular hybridization. RESULTS: Chip detection of serum specimens showed that 30 patients were HBV DNA positive and 10 HBV DNA negative in the 40 patients with HBV positive, 25 patients were HCV RNA positive and 15 patients were HCV RNA negative in the 40 patients with HCV positive, and all were HBV and HCV negative in the 40 healthy people. In 15 patients with HBV marker positive who were subjected to liver biopsy, 15 patients were detected HBV DNA positive in serum by gene chip, 15 patients HBcAg positive in liver tissues by immunocytochemistry, 14 patients HBV DNA positive in liver tissues by in situ molecular hybridization, and 14 patients HBV DNA positive in liver tissues by gene chip. Paraffin liver tissues specimens from the 99 patients with posthepatitis B cirrhosis showed that 67 patients were detected HBcAg positive by immunocytochemistry, 53 patients HBV DNA positive by in situ molecular hybridization, and 46 patients HBV DNA positive by gene chip. In the 46 patients, 40 patients were detected HBV DNA and HBcAg positive by in situ molecular hybridization and immunocytochemistry, 6 patients only HBcAg positive, and 33 patients HBcAg negative. CONCLUSIONS: The designed diagnostic gene chip can be used to simultaneously detect serum HBV DNA and HCV RNA, but the positive rate of HCV RNA diagnosed by this chip is lower. The gene chip can detect HBV DNA in serum and in liver tissue.

[Detection and identification of emergence of HBV YMDD motif mutation during lamivudine treatment by hybridization to an oligonucleotide array].

OBJECTIVE: Gene chip technology was set up to quickly and accurately detect and identify the HBV P gene YMDD motif mutation during the chronic hepatitis treated with lamivudine. METHODS: DNA microarrays were prepared by spotting fluorescence labeled probes of target genes onto specially lattice glass slides with robotics. The serum samples from 30 patients with hepatitis B after 68-week treatment with lamivudine were detected double-blind by gene chips and by nucleotide sequences assay technique to identify the rate of emergence of HBV P gene YMDD motif mutation. RESULTS: Twenty-one patients were found HBV P gene YMDD motif mutation by the gene chips including 11 cases with YVDD and 10 cases with YIDD motif mutation. By direct sequencing of the PCR products, 11 cases were found to have YVDD with adenine741 changed into cytidine resulted in methionine552 changed into valine in which 6 cases with adenine669 changed into cytidine and leucine changed into methionine, 10 cases had YIDD motif mutation with guanosine743 altered thymidine methionine552 changed into isoleucine, including 3 cases with thymidine281 changed into cytidine and leucine565 altered proline. CONCLUSION: The gene chip can be used to test HBV YVDD,YIDD motif mutation compared with nucleotide sequences assay technique, the accuracy rate was 100%.