Biomedical subjects

Masuo Aizawa

Publications and source records attributed to Masuo Aizawa.

24 records · Page 2Linked to original sources

PubMed2002

PKC-dependent IL-6 production and inhibition of IL-8 production by PKC activation in normal human skin fibroblasts under extremely high hydrostatic pressure.

Normal human dermal fibroblasts were found to survive and to be active in producing interleukin (IL)-6 and IL-8 under extremely high hydrostatic pressure, up to 40 MPa (1 atm=0.101325 MPa=1.03323 kgf/cm(2)), for 20 min. An inhibitor of protein kinase C (PKC) reduced the amount of IL-6 production, whereas IL-8 production was increased following pressure application. The activation of PKC in response to exposure to the pressure stress was detected by using the PKC-specific probe Rim-1. These findings indicate that IL-6 production induced by hydrostatic pressure stresses was dependent on the PKC signaling pathway. In contrast, pressure-induced IL-8 production was inhibited by PKC activity.

Catalytic Domain↗

PubMed2002

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status.

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.

Alcohols↗

PubMed2002

Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads.

Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell-free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild-type enzyme.

Cell-Free System↗

PubMed2002

Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid.

Atomic force microscopy (AFM) was used to analyze DNA bending induced by integration host factor (IHF). The direct AFM visualization of IHF-DNA complexes on the OP1 promoter regulatory regions on the Pseudomonas TOL plasmid showed that there was no intrinsic DNA bend in the OP1 promoter region, but a sharp DNA bend was induced by binding of IHF to the region between the upstream regulatory sequence and the promoter sequence. The DNA bending angles were distributed with a mean bend angle of 123 degrees. The IHF-DNA complexes were shown to bend at the IHF binding site giving rise to an asymmetric structure. These results provide direct evidence that IHF is required functionally for activation of OP1 transcription and support the DNA-loop model that the sharp DNA bend induced by binding of IHF facilitates the contact between RNA polymerase bound by the promoter sequence and XylR protein attached to the upstream sequence in the OP1 promoter.

Bacterial Proteins↗

PubMed2002

Cellular biosensing system for discovery of protein synthesis inhibitors with an electrochemical phosphate modulator to regulate the acid phosphatase gene expression of Saccharomyces cerevisiae.

A cellular biosensing system for screening protein synthesis inhibitors has been developed by linking an electrochemical phosphate modulator and matrix-immobilized yeast cells with an optical sensing device. To screen the protein synthesis inhibitors, yeast phosphatase gene regulating system has been employed by linking an electrochemical phosphate modulator. Since the yeast phosphatase gene coding gammaAPase is expressed, when the phosphate concentration in solution is lowered below the threshold, the gammaAPase production is triggered by lowering the phosphate concentration with the electrochemical phosphate modulator, and monitored continuously with the photometric device. The electrochemical phosphate modulator was assembled with matrix-immobilized yeast cells. The module could insert to ordinal cuvette to monitor the induced gammaAPase activity in an ordinal photometer. Using the system, induction profile of protein synthesis was easily observed and was affected remarkably by various protein synthesis inhibitors. This seems promising that the system can be applied for first screening process of de novo protein inhibitors. The cellular biosensing system seems promising in screening protein synthesis inhibitors.

Acid Phosphatase↗

PubMed2002

Post-transcriptional regulation of immunomodulatory cytokines production in human skin fibroblasts by intense mechanical stresses.

We found that extremely high hydrostatic pressure stresses induced a variety of cytokines production in normal human dermal fibroblasts. Normal human dermal fibroblasts were found to survive and were active in producing interleukin (IL) -6, -8, and monocyte chemoattractant protein-1 (MCP-1) under extremely high hydrostatic pressure, up to 70 MPa (=690.8 atm=713.8 kgf/cm2). 70 MPa pressure application extremely enhanced IL-6 and IL-8 secretions (about 130 folds) without transcriptional enhancement. Although induction of IL-1alpha, IL-1beta, and IL-12 mRNAs was appeared under high hydrostatic pressure condition, no translation of IL-1alpha, IL-1beta, and IL-12 proteins was found. Extracellular accumulation of constitutively produced MCP-1 was down regulated in the pressure-applied fibroblasts in the absence of transcriptional repression. These results indicated that hydrostatic pressure stresses triggered post-transcriptional regulation mechanisms that modulated the cytokines production.

Journal Article↗