Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.     

Laser and electrochemical complex machining of micro-stent with on-machine three-dimensional measurement

In the micro-fabrication, small installation misalignment and deformation of the workpiece cause fatal damage. On-machine measurement is one of the efficient methods to prevent this damage. Our system performs the laser machining and the three-dimensional measurement of the workpiece position using only one laser. Based on the measurement result, the position error of the workpiece is automatically corrected and laser-machined. Electrochemical machining (ECM) is also done on the same machining system. In this paper, the configuration of the system is explained. Measurement and error correcting techniques are described in detail. And the experimental results that made stent shape using the micro-tube of diameter 120 μm and 210 μm are shown.     

The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations

Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3′→5′ exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.     

Observation/confirmation of hindered E2 strength in 18C/16C

We have measured the lifetime of the first excited 2⁺ state in ¹⁸C using an upgraded recoil shadow method to determine the electric quadrupole transition. The measured mean lifetime is 18.9±0.9 (stat)±4.4 (syst) ps, corresponding to B(E2;2₁ ⁺ $ \rightarrow$ 0⁺ _gs) = 4.3±0.2±1.0e ² fm^4, or about 1.5 Weisskopf units. The mean lifetime of the first 2⁺ state in ¹⁶C was remeasured to be 18.3±1.4±4.8 ps, about four times shorter than the value reported previously. The discrepancy is explained by incorporating the $ \gamma$ -ray angular distribution obtained in this work into the previous measurement. The observed transition strengths in ^{16, 18}C are hindered compared to the empirical values, indicating that the anomalous E2 strength observed in ¹⁶C persists in ¹⁸C .     

Novel internal target for electron scattering off unstable nuclei

A novel internal target has been developed, which will make electron scattering off short-lived radioactive nuclei possible in an electron storage ring. An "ion trapping" phenomenon in the electron storage ring was successfully utilized for the first time to form the target for electron scattering. Approximately 7 x 10(6) stable 133Cs ions were trapped along the electron beam axis for 85 ms at an electron beam current of 80 mA. The collision luminosity between the stored electrons and trapped Cs ions was determined to be 2.4(8) x 10(25) cm(-2) s(-1) by measuring elastically scattered electrons.     

Photochemically induced coupling reaction of triarylstibines with olefins

Photoreaction of triarylstibines with styrenes resulted in the formation of 2-aryl-1-phenylethanols accompanied by air oxidation. Formation of the products has been explained by the valence expansion of the oxygen-antimony-styrene complex to a five-valent intermediate, followed by reductive coupling.     

Resonance Raman and luminescence studies on the quasi-one dimensional crystals of [Pt(en)z][Pt(en)2Cl2](ClO4)4

Resonance Raman scattering, infrared luminescence and absorption spectra of [Pt(en)₂][Pt(en)₂Cl₂](ClO₄)₄ single crystals have been measured for polarized light at 2 K and room temperature. Contrary to the previous works on polycrystals, the incident photon energy dependence of the Raman cross section due to the symmetric ClPt^IVCl stretching mode has a resonance peak at the charge-transfer absorption edge, which is well explained with the two-band model in a one-dimensional system. Two characteristic structures observed below the absorption edge are concluded not to be related to the charge-transfer transition by measurements of the excitation spectra both of the Raman scattering and of the luminescence. The origin of this luminescence is also discussed.     

Regioselective conjugation of chitosan with a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg, and evaluation of its inhibitory effect on experimental cancer metastasis

A conjugate from the YIGSR peptide and chitosan has been prepared on the basis of a regioselective modification strategy of chitosan, and its antimetastatic activity has been assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled with the peptide portion containing a spacer amino acid, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-OH [beta Ala; beta-alanine]. The product was treated with CHCl2CO2H to afford the desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-chitosan, which proved to inhibit the experimental lung metastasis of B16BL6 melanoma cells in mice at lower doses than the parent peptide.     

Polyaddition of bisketenimines. II. Synthesis of poly(N-acylamides) from bisketenimines and dicarboxylic acids

Poly(N-acylamides) were synthesized by polyaddition of bisketenimines with dicarboxylic acids. Yields and inherent viscosities of these polymers were low, presumably because of side reactions. The thermal stability and solubility of the polymers were studied.