Biomedical subjects
Publications and source records attributed to Yoshio Kato.
A linear, covalently-closed, dumbbell-shaped DNA vector including a transcription unit is known to have both biological stability and safety and is expected to be useful for gene therapy. We established an easy, quick, and large preparative synthetic method of modified- and unmodified-dumbbell DNA using an intramolecular cyclization at the DNA termini.
Eukaryotic tRNAs are transcribed, processed and exported from the nucleus to the cytoplasm to participate in protein biosynthesis. A variant of human gene for tRNA(Val), HtV4, is not processed neither its 5'- nor its 3'-flanking sequences. By RT-PCR analysis and in situ hybridization, we demonstrated that the HtV4 was constitutively expressed without processing, and localized in the nucleus in HeLa cells.
The basic properties of a new dye affinity adsorbent Toyopearl AF-Blue HC-650M and its applications to the purification of proteins were studied. The binding capacity for human serum albumin (HSA) was greater than 18 mg per ml gel. The dye leakage from Toyopearl AF-Blue HC-650M in 0.5 M NaOH and 0.5 M HCI was less compared with an agarose adsorbent. Caustic stability study also demonstrated this material withstood exposure to 0.1 M NaOH for 1 month with no significant loss of binding capacity for HSA. We purified human albumin from human serum and lactate dehydrogenase (LDH) from rabbit muscle extract in a single step. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that human albumin and LDH were highly purified.
We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.
To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.