Improvement of translation efficiency in an Escherichia coli cell-free protein system using cysteine.

Cell-free protein synthesis systems are powerful tools for protein expression, and allow large amounts of specific proteins to be obtained even if these proteins are detrimental to cell survival. In this report we describe the effect of cysteine on cell-free protein synthesis. The addition of cysteine caused a 2.7-fold increase in the level of synthesized glutathione S-transferase (GST). Moreover, the levels of sulfhydryl group reductants, including reduced glutathione and dithiothreitol (DTT), were increased 1.9- and 1.7-fold, respectively, whereas levels of the disulfide dimers, cystine and oxidized glutathione, were suppressed 87% and 66%, respectively. These trends were also observed for green fluorescent protein (GFP) expression. The addition of cysteine competitively reversed the inhibitory effect of cystine on protein expression. These results suggest that the sulfhydryl group in cysteine plays a crucial role in enhancing protein synthesis, and that the addition of excess cysteine could be a convenient and useful method for improving protein expression.

Methylene chain ruler for evaluating the regioselectivity of a substrate-recognising oxidation catalyst.

Regioselective C-H oxidation of aliphatic molecules with synthetic catalysts is challenging. We incorporated substrate-recognition sites into a ruthenium porphyrin-heteroaromatic N-oxide catalytic system in order to characterise its regioselectivity for the oxidation of alkanes. This substrate-recognition catalytic reaction exhibits high regioselectivity and high reaction efficiency.

Nano-kinetics of probe-particles in solution visualized by a pin-fiber video scope.

The nano-kinetics of colloidal particles and living cells with the colloidal particles were visualized by a newly developed video scope. The system of the new video scope has a feature of fine controlling the illumination conditions by using a single optical fiber. This characteristic enables one to obtain clear images of living cells and the motions of colloidal particles by light-scattering effects. In the experiments, RBL-2H3 cells and gold colloidal particles were observed. Scattering images with high contrast and a dark background like in dark-field observations could be attained. In the experiments, a pulsed laser was also applied. The results obtained in this study could validate the effectiveness and possibility of a new video scope for applications to biological and biomedical fields.

Development of the single-cell MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass-spectroscopic assay.

MALDI-TOF mass spectrometry was used to detect intracellular molecules from a single intact cell on monolayers of other cells. Intracellular molecules, e.g., histamine, were gradually increased in a mouse bone marrow-derived mast cell by a maturation process. A single cell was captured by a microsuction pipette, and the mass spectra of intracellular histamine were measured directly. Finally, the time course of the intracellular molecular contents and the maturation stage from a single cell were estimated.

Analysis of the nano-kinetic movement of a single DNA by a pin-fiber video scope.

The nano-kinetics movement of a single DNA molecule was visualized by a newly developed video-microscope system with an optical fiber, called a "pin-fiber video scope". The stretching and shrinking motion was clearly observed, and its kinetics was analyzed by numerical calculations. The new video-microscope system has a potential to analyze the nano-kinetics of a molecule.

[An example of self-evaluation of a sense of achievement by students in 6-year pharmacy school with the model core curriculum of pharmaceutical education].

In March 2012, the first students, finishing the newly introduced 6-year-course of pharmaceutical education, have graduated and gone out into the world. At this point, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) is going to revise the model core curriculum of pharmaceutical education to be more suited for educating students to achieve their goal of becoming the clinical pharmacist standard defined by the revised School Education Act. Here we report the self-evaluation study based on the survey using questionnaire about a sense of achievement with Visual Analog Scales, regarding the fundamental quality as a pharmacist standard proposed by the Professional Activities Committee in the MEXT. The sample size of survey was about 600 of students studying in the Faculty of Pharmaceutical Sciences in Josai International University (JIU) and the survey was carried out during the period of March-April in 2012. The study suggested that the majority of graduates were satisfied with the new education system and marked as a well-balanced quality to be a pharmacist standard, after completing the 6-year pharmaceutical education based on "the model core-curriculum". It would be worthwhile to perform this kind of survey continuously to monitor the student's self-evaluation of a sense of achievement to verify the effectiveness of 6-year-course pharmaceutical education based on the newly establishing core curriculum in Japan.

Observation and analysis of single DNA nano-kinetics by pin-fiber video scope.

The nano-kinetic movement of a single DNA molecule was observed and analyzed by a newly developed video-microscope system with an optical fiber, called a pin-fiber video scope. A single lambda-DNA molecule was put in focus using fiber-illumination, and the stretching and shrinking motion was measured. The molecule's kinetics were analyzed by numerical calculations and are discussed. A photocleavage phenomenon of DNA molecules was also visualized by the pin-fiber video scope. The new video-microscope system has the potential to observe and analyze the nano-kinetics of a single molecule.