Biomedical subjects

Shohei Yamamura

Publications and source records attributed to Shohei Yamamura.

5 recordsLinked to original sources

PubMed2006

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications.

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

DNA↗

PubMed2005

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Amino Acid Sequence↗

PubMed2002

Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp.

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin.

Bacterial Proteins↗

PubMed2002

Characterization of a new keratin-degrading bacterium isolated from deer fur.

A keratin-degrading bacterium was isolated from soil containing deer fur. An axenic culture of the keratin-degrading bacterium was obtained in liquid culture using a keratin enrichment technique. The isolated bacterium was gram negative and catalase- and oxidase-positive. Transmission electron microscopic observations showed that the bacterium was rod-shaped, 1.0-1.3 microm long and 0.7 microm in diameter. Phylogenetic analysis of 16S rDNA revealed that the new isolate has only 90.6% homology with Stenotrophomonas nitritireducens. Hence, this new bacterium was designated as Stenotrophomonas sp. D-1. The optimum temperature was determined to be 20 degrees C for maximum growth and keratinolytic enzyme production. Amino acid data, obtained after treating keratin powder with the supernatant culture, suggest that the major free amino acids resulting from keratin degradation are phenylalanine, tyrosine and valine. In addition, native chicken feather was degraded completely at 20 degrees C in 2.5 d by this bacterium.

Journal Article↗

PubMedDate not supplied

Selection and properties for the recognition of P19 embryonic carcinoma stem cells.

The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.

Animals↗