Shigeo Tsuchiya

Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.

We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.

Isothermal RNA Sequence Amplification Method for Rapid Antituberculosis Drug Susceptibility Testing of Mycobacterium tuberculosis

ABSTRACT RNA transcript quantification by an isothermal sequence amplification reaction was evaluated for susceptibility testing of 15 Mycobacterium tuberculosis strains. Agreement with the proportion method on Ogawa egg medium and the BACTEC MGIT 960 system was 100 and 87% for rifampin, 93 and 100% for isoniazid, 60 and 53% for ethambutol, and 80 and 80% for streptomycin, respectively.

Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker

FP6, a novel recombinant fusion protein of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), was prepared in the methylotrophic yeast Pichia pastoris. This protein was a potent activator of a cell surface transducing glycoprotein, gp130 and is a potential therapeutical reagent in the hemopoietic field. A linker is generally thought to be required for two fused molecules to retain their proper structures although it should preferably be removed to reduce possible antigenicity. It was found that the C-terminal residue of IL-6R could be directly linked to the N-terminal residue of IL-6 without decreasing the ability of IL-6 to bind gp130 and send the IL-6 signal. It was also found that the peptide bond between Lys-37 and Asp-38 of IL-6 was prone to proteolytic cleavage and that the immunoglobulin (Ig)-like region of IL-6R underwent extensive and heterogeneous glycosylation when expressed in P. pastoris. Based on these findings, we designed FP6 without the Ig-like region, in which the C-terminal residue of Ala-333 of IL-6R was directly linked to Asp-38 of IL-6 by a peptide bond. Purified FP6 had both an in vitro effect on hemopoietic progenitors to generate various colonies and an in vivo effect on megakaryocyte progenitors to increase platelet counts. Four purified FP6s were obtained, which had the same molecular mass and different isoelectric points without any detectable modification in the course of purification. The difference in isoelectric points was shown to be due to microheterogeneity of the carbohydrate chains. Each FP6 had the same specific activity in the cell growth assay with or without endoglycosidase digestion. Homogeneous FP6 with respect to isoelectric point as well as molecular mass merits more detailed characterization and evaluation for possible clinical application.