Mitsuru Niwano

Antioxidant activity of tannins and flavonoids in Eucalyptus rostrata

Abstract Antioxidant activity of an extract from Eucalyptus rostrata was the highest among 16 Eucalyptus species tested. From the leaves of Eucalyptus rostrata nine active compounds were isolated: 1,2,6-tri- O -galloyl-β- d -glucose and tellimagrandin 1 as tannins; spiraeoside, hyperoside, isoquercitrin, and myricetin 3- O -α- l -arabinopyranoside as flavonol glycosides; quercetin 3- O -α-arabinopyranoside-2″-gallate, kaempferol 3- O -α-arabinopyranoside-2″-gallate, quercetin 4′- O -β- d -glucopyranoside-6″-gallate as acylated flavonol glycosides. Antioxidant activity of the tannins and acylated flavonol glycosides, all with galloyl groups, was much higher than that of a synthetic antioxidant. Two of the compounds, spiraeoside and quercetin 4′-glucosylgallate, showed high superoxide dismutase activity.

Immunosuppressive activity of a monoterpene from Eucommia ulmoides

Abstract Loliolide has been isolated from the chloroform extract of the leaves of Eucommia ulmoides and shown to have immunosuppressive activity.

Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system

Detailed analysis of cell-free translation, coupled transcription-translation in static conditions and a continuous flow system based on E. coli S30 extracts was performed. Degradation of template mRNA was the predominant trigger to terminate the protein synthesis. In a coupled system, mRNA was preserved by repeated transcription whereas the starvation of nucleotide triphosphates led to the termination of protein synthesis in less than 1 h. In the CFCF system, NTP was held at the level of initial concentration and therefore did not arrest the translation for 15 h. The accurate coupling of transcriptional rate and translational rate was also crucial to enhance the efficiency of protein synthesis.

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

Abstract heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.

Utilization of copper ion as a ribonuclease inhibitor in a cell-free protein synthesis system

Abstract The degradation of mRNA in a cell-free coupled transcription and translation system derived from Escherichia coli was prevented by the addition of 0.5 mM copper ion. Neither the transcription nor the translation was influenced by the presence of copper ion at this concentration, whereas the transcription was inhibited by a copper ion concentration exceeding 1.0 mM, and the translation by a concentration of 1.0 mM or more. In the cell-free system conducted with mRNA as a template, the amount of protein synthesized increased markedly in the presence of 0.5 mM copper ion. These results indicate that copper ion can stabilize cell-free protein synthesis systems by preventing mRNA degradation.

Two acylated flavonol glycosides from Eucalyptus rostrata

Abstract From the leaves of Eucalyptus rostrata, two new acylated flavonol glycosides were isolated together with quercetin 3-O-α-arabinopyranoside-2″-gallate and quercetin 4′-O-β- d -glucopyranoside. The structures of the new compounds were identified as quercetin 4′-O-β- d -glucopyranoside-6″-gallate and kaempferol 3-O-α-arabinopyranoside-2″-gallate.

Enhancement of protein synthesis in continuous-flow, cell-free system by improvement of membrane permeation

Abstract The productivity of the continous-flow, cell-free protein synthesis system (CFCF-system) is inferior compared to that in the cell-free batch system. By comparing the synthesized protein present between in the reaction chamber and in the fed-out solution in the CFCF-system, it appeared that much of the synthesized protein remained in the reaction chamber. Addition of a nonionic detergent, n -dodecyl β- d -maltoside, to the feeding solution resulted in enhancement of the productivity in the CFCF-system which indicated that the permeability of the membrane to the protein might be one of the most influenctial factors affecting the productivity of the CFCF-system.

Improvement of Escherichia coli cell-free system by utilization of cell extract having additional property. Problems and countermeasures.

In theEscherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E.coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.