Simultaneous analysis of major ingredients of Gardenia fruit by HPLC-MS/TQMS method

An efficient, accurate HPLC-MS/TQMS method was introduced for the quantitative/qualitative simultaneous analysis of main ingredients, namely geniposide and genipingentiobioside, in the Gardenia fruit. The separation was successfully obtained using a C8 (100mm×2.1mm, 5μm, 30°C) column by gradient elution with ultrapure water as mobile phase, where flow rate was set to 0.2 ml/min and detection wavelength at 240 nm. The analytical method was validated and the quantification of active compounds, namely genipingentiobioside and gardenoside, was performed. Linearity, precision, repeatability, stability and recovery were also reported. The quantitative analysis revealed that both main ingredients as geniposide and genipingentiobioside have performed a good linear relationship in 0.1-100 mg/ml concentration range (r=1.00000 and r =0.99998). The average content was measured to be 4.842% with RSD 0.96% for geniposide and 1.1976% with RSD 0.47% for genipingentiobioside in the Gardenia fruit. Accordingly, this method would be feasible for the quantity and quality control of crude drugs

EXPRESSION OF BACTERIOOPSIN GENES IN ESCHERICHIA COLI

An inducible expression vector pUBO was constructed with native codons in order to express the gene of Bacteriorhodopsin (BOP) in Escherichia coli (E. coli). Vector pUBO contains lac-promoter followed by the partial structural gene of lacZ and the structural gene of BOP. The expression of this fusion protein was detected by ELISA with anti-BOP antiserum. The fusion protein obtained from E. coli trnsformed with pUBO formed approximately 0.1% of the total protein of the E. coli membrane fraction

LIGHT-DEPENDENT PROTON MOVEMENT AND PHOTOINTERMEDIATES ON BACTERIORHODOPSIN PHOTOCYCLE

The relationship between photointermeidates of bacteriorhbdopsin (bR) and proton uptake was investigated by a flash photolysis technique. Attention was paid to keep the sample in a light-adapted state and under the same conditions during measurements. The decay of M-intermediate (M), the formation and decay of O-intermediates (O), the recovery of bR and release and the uptake of protons were measured with a purple membrane suspension (pH 7.0, 0.5 M NaCl) at various temperatures. The following reaction scheme for the bR photocycle was deduced from the results. 1) The decay process of M consists of three components (M_, M_ and M_s). 2) O is formed from the faster component of M (M_). 3) A part of M (M_ and M_s) decays directly to bR without passing through O. 4) The uptake of protons is coupled with the decay of the slowest M (M_s). The above reaction scheme was confirmed by the results obtained from similar experiments using an analog pigment of bR, Np-bR, M of which decays much more slowly than that in native bR system

Low-temperature photoreaction cycle of phoborhodopsin (sensory rhodopsin II) from Halobacterium salinarium

There are four types of retinal proteins in the membrane of Halobacterium salinarum. Bacteriorhodopsin (BR) and halorhodopsin (HR) convert light energy into electrostatic gradients that can be used by the cell as an energy source to produce ATP. Sensory rhodopsin I (SR I) and phoborhodopsin (pR) enable the cells to migrate toward an environment optimal for light energy harvesting while avoiding potentially damaging shorter wavelength light. In the present work, low temperature photoreaction cycle of pR expressed in E. coli was studied. Comparing to the previous results, the new findings in the present work are: (i) The K-like intermediate was found to be a mixture of two photoproducts. (ii) Formation of an L-like intermediate (P482) was observed. (iii) Upon light irradiation, formation of a long lived shorter wavelength photoproduct (P370) was observed at 20 ℃.特集 : 「資源、新エネルギー、環境、防災研究国際セミナー

Expression of Partial Genes of Bacterioopsin in Escherichia Coli

Bacteriorhodopshin (BR) is an intrinsic membrane protein composed of seven membrane-spanning helices (A-G). Partial genes of bacterioopsin (BOP) which encode the peptides including ABCD helices and EFG helices of bacteriorhodopsin were independently expressed in Escherichia coli (E. coli?). Six inducible expression vectors were constructed, three of which contain a partial gene coding the ABCD helices of BOP (vectors pUBOAIN, pKBOAIN and pTKBOAIN), and three coding the EFG helices of BOP (vectors pUBOAIC, pKBOAIC and pTKBOAIC). The vectors pUBOAIN and pUBOAIC contain lac-promoter and the vectors pKBOAIN and pKBOAIC contain tac-promoter followed by the partial genes of BR. The vectors pTKBOAIN and pTKBOAIC contain a nucleotide fragment encoding the presequence of the manganese-stabilization protein of Anacystis nidurans between the lac-promoter and the BR partial gene. The expression of the resulting fusion proteins were detected by ELISA using mouse anti-BR serum. The fusion proteins prepared from F. coli transformed by pTKBOAIN or pTKBOAIC were estimated to comprise more than 1% of the total membrane protein in the E. coli

Analysis of Surface Acoustic Wave Propagation Velocity in Biological Function-Oriented Odor Sensor

We describe the measurement of surface acoustic wave (SAW) velocities to identify five odorant molecules by using a SAW device system. We derive a new frequency equation for SAWs propagating in the SAW device system with Cynops pyrrhogaster lipocalin (Cp-Lip1) protein, in order to identify five odorant molecules, R-limonene (R-Lim), ethyl butyrate (Eth), 2-isobutylthiazole (Iso), benzophenone (Ben), and 2-acetylthiazole (Ace). We developed a method to identify these odorant molecules combined with the Cp-Lip1 odorant-binding protein. Our frequency equation can satisfactorily predict different odorant molecules in the Cp-Lip1 SAW device. Moreover, our data suggest that the propagation velocity of the SAWs mostly relate to the density and concentration of the Cp-Lip1 odorant molecule mixtures. At the same sample concentration, the propagation velocity depends on the density. For the same odorant molecule, the propagation velocity decreases with increasing concentration