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Biomedical subjects

Shinsuke Nakade

Publications and source records attributed to Shinsuke Nakade.

3 recordsLinked to original sources

Quantitative detection system for maize sample containing combined-trait genetically modified maize.

Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.

DNA, Plant↗

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.

Base Sequence↗

Characterization of HscC (Hsc62), homologue of Hsp70 in Escherichia coli: over-expression of HscC modulates the activity of house keeping sigma factor sigma70.

BACKGROUND: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with sigma32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown. RESULTS: We observed that the over-expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a beta-galactosidase assay and found that HscC reduced the sigma70-dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited sigma70-dependent transcription. In addition, the co-purification analysis showed that sigma70 co-eluted with HscC. CONCLUSION: These results indicate that HscC forms a complex with sigma70 and may function as its negative modulator.

DNA-Directed RNA Polymerases↗