Publications by authors named "Yoichi Shibusawa"

27 Publications

  • Page 1 of 1

Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

J Pharm Biomed Anal 2016 Sep 8;128:455-461. Epub 2016 Jun 8.

Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan. Electronic address:

Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the β-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanol
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http://dx.doi.org/10.1016/j.jpba.2016.06.009DOI Listing
September 2016

Reversed-phase liquid chromatographic analysis of hydrophobic interaction between proanthocyanidins and a C₈-alkyl compound in aqueous solution.

Biosci Biotechnol Biochem 2016 16;80(3):419-25. Epub 2015 Nov 16.

a Division of Pharmaceutical and Biomedical Analysis , School of Pharmacy, Tokyo University of Pharmacy and Life Sciences , Hachioji , Japan.

Structural and physicochemical properties of oligomeric flavan-3-ols (proanthocyanidins) in aqueous solution were investigated by spectrometric and reversed-phase (RP) HPLC analyses. Circular dichroism and fluorescence spectra of (-)-epicatechin (EC) oligomers linked through C-4 to C-8 interflavan bonds showed that EC oligomers larger than dimers formed a stable secondary structure in water. These EC oligomers are water-soluble hydrophilic compounds, whereas the oligomers were strongly retained by a C8-alkyl stationary phase under conventional RP-HPLC conditions. In a further C8-HPLC study, the hydrophobic interaction between EC oligomers and 1-octanesulfonic acid sodium salt (OSA Na) added to the mobile phase was quantitatively evaluated based on the relationship between the logarithm of the retention factor of the solute and the OSA Na concentration in the mobile phase. The strength values of the hydrophobic interaction of EC oligomers larger than dimers were the highest of 22 tested polyphenolic standards.
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http://dx.doi.org/10.1080/09168451.2015.1107465DOI Listing
October 2016

High-throughput determination of octanol/water partition coefficients using a shake-flask method and novel two-phase solvent system.

J Pharm Biomed Anal 2016 Jan 25;117:338-44. Epub 2015 Sep 25.

Division of Pharmaceutical and Biomedical Analysis, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Electronic address:

A high-throughput method for determining the octanol/water partition coefficient (P(o/w)) of a large variety of compounds exhibiting a wide range in hydrophobicity was established. The method combines a simple shake-flask method with a novel two-phase solvent system comprising an acetonitrile-phosphate buffer (0.1 M, pH 7.4)-1-octanol (25:25:4, v/v/v; AN system). The AN system partition coefficients (K(AN)) of 51 standard compounds for which log P(o/w) (at pH 7.4; log D) values had been reported were determined by single two-phase partitioning in test tubes, followed by measurement of the solute concentration in both phases using an automatic flow injection-ultraviolet detection system. The log K(AN) values were closely related to reported log D values, and the relationship could be expressed by the following linear regression equation: log D=2.8630 log K(AN) -0.1497(n=51). The relationship reveals that log D values (+8 to -8) for a large variety of highly hydrophobic and/or hydrophilic compounds can be estimated indirectly from the narrow range of log K(AN) values (+3 to -3) determined using the present method. Furthermore, log K(AN) values for highly polar compounds for which no log D values have been reported, such as amino acids, peptides, proteins, nucleosides, and nucleotides, can be estimated using the present method. The wide-ranging log D values (+5.9 to -7.5) of these molecules were estimated for the first time from their log K(AN) values and the above regression equation.
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http://dx.doi.org/10.1016/j.jpba.2015.09.019DOI Listing
January 2016

Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy.

J Pharm Biomed Anal 2014 Jul 20;95:47-53. Epub 2014 Feb 20.

Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan. Electronic address:

Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.
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http://dx.doi.org/10.1016/j.jpba.2014.02.009DOI Listing
July 2014

Comprehensive separation and structural analyses of polyphenols and related compounds from bracts of hops (Humulus lupulus L.).

J Agric Food Chem 2014 Mar 26;62(10):2198-206. Epub 2014 Feb 26.

Research Laboratories for Fundamental Technology of Food, Asahi Group Holdings, Limited, 1-21, Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan.

A novel sequential chromatographic technique was applied to the comprehensive separation of polyphenols and related compounds from a hop bract extract. Over 100 types of constituents were effectively isolated from only 25 g of extract in high yields by high-speed countercurrent chromatography followed by hydrophilic interaction chromatography and reversed-phase high performance liquid chromatography. Among the materials isolated, the structures of 39 compounds were elucidated on the basis of their spectroscopic data including electrospray ionization time-of-flight mass spectrometry and one-dimensional/two-dimensional nuclear magnetic resonance. Three new compounds, 1 known compound identified for the first time in plants, and 20 known compounds that have not been reported in hops, were found. The hop bract extract also contained an abundance of highly oligomeric proanthocyanidins, which consisted of B-type procyanidin structures.
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http://dx.doi.org/10.1021/jf405544nDOI Listing
March 2014

Separation of nucleobases and their derivatives with organic-high ionic strength aqueous phase systems by spiral high-speed counter-current chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2012 Apr 16;891-892:94-7. Epub 2012 Feb 16.

Division of Pharmaceutical and Biomedical Analysis, School of Pharmacy, Tokyo University of Pharmacy and Life sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan.

A set of nucleic acid constituents were separated with ultra polar two-phase solvent systems by a spiral multilayer coil mounted on the rotary frame of a type-J coil planet centrifuge. These two-phase systems were composed of 1-butanol/ethanol/50% saturated aqueous ammonium sulfate at various volume ratios. Nucleobases including adenine, cytosine, uracil, and thymine; nucleosides including adenosine, guanosine, cytidine, and uridine; and nucleotides including, AMP, GMP, CMP, UMP, and TMP are partitioned in each group with suitable solvent ratios. Adenine derivatives such as adenosine, AMP, ADP, and ATP were well resolved in the most polar solvent system composed of ethanol/50% saturated aqueous ammonium sulfate at a volume ratio of 1:2. It was found that cytosine and cytidine peaks showed some irregular two peaks probably due to their keto and enol isomers, while the separation of AMP forms two peaks especially when TMP was added in the sample solution, the mechanism of which is now under investigation in our laboratory.
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http://dx.doi.org/10.1016/j.jchromb.2012.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311770PMC
April 2012

Counter-Current Chromatographic Separation of Nucleic Acid Constituents with a Polar Volatile Organic-Aqueous Two-Phase Solvent Systems with ELSD Detection.

Open Anal Chem J 2012 ;6:9-14

Bioseparation Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Highly hydrophilic volatile organic/aqueous two-phase solvent systems containing an organic salt such as, acetonitrile/800 mM and 1200 mM ammonium acetate (1 : 1, v/v) were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate hydrophilic compounds. The retention of the upper and the lower stationary phases in the column of the cross-axis coil planet centrifuge (CCC instrument) was studied by changing the flow-rate of the mobile phase (1.0-3.0 ml/min). Using the acetonitrile/800 mM ammonium acetate two-phase solvent system, the stationary phase was retained at 46.3% relative to the total column capacity of 65 ml by the reversed-phase elution mode at a flow-rate of 1.0 ml/min. The best retention of the stationary upper phase of 51.5% was obtained by the solvent system of the acetonitrile/1200 mM ammonium acetate at the above flow-rate. With the acetonitrile/800 mM ammonium acetate system the base line separation of adenine and adenosine monophosphate (AMP) detected by evaporative light scattering detector (ELSD) and UV was achieved with lower phase mobile at a flow-rate of 1.0 ml/min within 70 min.
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http://dx.doi.org/10.2174/1874065001206010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952559PMC
January 2012

An effective pulse sequence for detecting a ligand binding with a protein receptor using a WET sequence and the repeated Z-filters.

Anal Sci 2010 ;26(10):1107-10

Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan.

WaterLOGSY and STD experiments are widely used as NMR-based screening techniques in drug research. In the present study, an improved STD pulse sequence was developed, and its efficiency and applicability of observing the ligand signals were evaluated compared with the WaterLOGSY experiment. A combination of presaturation, a WET sequence and subsequent repeated Z-filters can provide the most effective water suppression, which is incorporated into the STD pulse sequence. In a sample solution of tryptophan and glucose in the presence of human serum albumin, the improved STD experiment only succeeded in selective detections of the bound ligand signals, even resonating close to water.
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http://dx.doi.org/10.2116/analsci.26.1107DOI Listing
February 2011

Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system.

J Chromatogr A 2010 May 16;1217(20):3457-60. Epub 2010 Mar 16.

Division of Pharmaceutical and Biomedical Analysis, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using type J coil planet centrifuge. The separation was performed with a hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1:1, v/v) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight selected nucleic acid constituents (4.0 mg, 0.5 mg of each), uridine monophosphate (UMP), adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), uridine, urasile, deoxy uridine, adenosine and adenine were well resolved within 160 min.
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http://dx.doi.org/10.1016/j.chroma.2010.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860025PMC
May 2010

A reusable liposome array and its application to assay of growth-hormone-related peptides.

Anal Bioanal Chem 2010 Jun 20;397(3):1377-81. Epub 2010 Mar 20.

Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan.

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 x 10(-9) to 0.5 x 10(-7) g/mL with detection limits of 2 x 10(-10) and 3 x 10(-10) g/mL, respectively.
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http://dx.doi.org/10.1007/s00216-010-3615-xDOI Listing
June 2010

Application of WET sequence for the detection of the ligand signals resonating close to water.

Magn Reson Chem 2009 Nov;47(11):971-6

Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

An efficient pulse sequence for observing the ligand signals resonating close to the water signal has been developed by incorporating the WET technique into the saturation transfer difference pulse sequence. Although several pulse sequences have been developed for observing a ligand binding with a protein receptor, the ligand signals resonating close to the water were undetectable owing to the interference of the huge water signal in the samples containing 95% (1)H(2)O. On the point of sample preparation, it is preferable to avoid the solvent exchange in the protein samples. In the proposed pulse sequence, a WET sequence is incorporated for the selective suppression of the water resonance. The efficient water suppression and the clear observation of the bound ligand signals close to the water have been demonstrated using the lysozyme-glucose complex.
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http://dx.doi.org/10.1002/mrc.2493DOI Listing
November 2009

Counter-current chromatographic estimation of hydrophobicity of Z-(cis) and E-(trans) enalapril and kinetics of cis/trans isomerization.

J Chromatogr A 2007 Jul 19;1157(1-2):101-7. Epub 2007 Apr 19.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The kinetics of Z-(cis)/E-(trans) isomerization of enalapril was investigated by reversed phase high-performance liquid chromatography (RP-HPLC) using a monolith ODS column under a series of different temperature and pH conditions. At a neutral pH 7, the rate (k(obs)) of Z-(cis)/E-(trans) isomerization of enalapril at 4 degrees C (9.4 x 10(-3)min(-1)) is much lower than at 23 degrees C (1.8 x 10(-1)min(-1)), while the fractional concentration of Z-(cis) isomer is always higher than that of E-(trans) isomer in the pH range 2-7. The fractional concentration of the E-(trans) isomer becomes a maximum (about 40%) in the pH range 3-6, where enalapril exists as a zwitterion. The hydrophobicity (logP(O/W)) of both isomers was estimated by high-speed counter-current chromatography (HSCCC). Normal phase HSCCC separation using a tert-butyl methyl ether-acetonitrile-20mM potassium phosphate buffer (pH 5) two-phase solvent system (2:2:3, v/v/v) at 4 degrees C was effective in partially separating the isomers, and the partition coefficient (K) of each isomer was directly calculated from the retention volume (V(R)). The logP(O/W) values of Z-(cis) and E-(trans) isomers were -0.46 and -0.65, respectively.
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http://dx.doi.org/10.1016/j.chroma.2007.04.027DOI Listing
July 2007

Comprehensive separation of secondary metabolites in natural products by high-speed counter-current chromatography using a three-phase solvent system.

J Chromatogr A 2007 Jun 23;1151(1-2):74-81. Epub 2007 Mar 23.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

High-speed counter-current chromatography (HSCCC) using the three-phase solvent system n-hexane-methyl acetate-acetonitrile-water at a volume ratio of 4:4:3:4 was applied to the comprehensive separation of secondary metabolites in several natural product extracts. A wide variety of secondary metabolites in each natural product was effectively extracted with the three-phase solvent system, and the filtered extract was directly submitted to the HSCCC separation using the same three-phase system. In the HSCCC profiles of crude natural drugs listed in the Japanese Pharmacopoeia, several physiologically active compounds were clearly separated from other components in the extracts. The HSCCC profiles of several tea products, each manufactured by a different process, clearly showed their compositional difference in main compounds such as catechins, caffeine, and pigments. These HSCCC profiles also provide useful information about hydrophobic diversity of whole components present in each natural product.
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http://dx.doi.org/10.1016/j.chroma.2007.03.071DOI Listing
June 2007

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system.

J Chromatogr A 2007 Jun 3;1151(1-2):158-63. Epub 2007 Feb 3.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.
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http://dx.doi.org/10.1016/j.chroma.2007.01.111DOI Listing
June 2007

Retention behavior of oligomeric proanthocyanidins in hydrophilic interaction chromatography.

J Chromatogr A 2007 Mar 16;1143(1-2):153-61. Epub 2007 Jan 16.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

A novel method was developed for the separation of proanthocyanidins (PAs; oligomeric flavan-3-ols) by hydrophilic interaction chromatography (HILIC) using an amide-silica column eluting with an aqueous acetonitrile mobile phase. The best separation was achieved with a linear gradient elution of acetonitrile-water at ratios of 9:1 to 5:5 (v/v) for 60 min at a flow rate of 1.0 ml/min. Under these HPLC conditions, a mixture of natural oligomeric PAs (from apple) was separated according to degree of polymerization (DP) up to decamers. The DP of each separated oligomer was confirmed by LC/electrospray ionization MS. In further HILIC separation studies of 15 different flavan-3-ol and oligomeric PA (up to pentamer) standards with an isocratic elution of acetonitrile-water (84:16), a high correlation was observed between the logarithm of retention factors (log k) and the number of hydroxyl groups in their structures. The coefficient of this correlation (r2=0.9501) was larger than the coefficient (r2=0.7949) obtained from the correlation between log k and log P(o/w) values. These data reveal that two effects, i.e. hydrogen bonding between the carbamoyl terminal on the column and the hydroxyl group of solute oligomer and hydrophilicity based on the high-order structure of oligomeric PAs, corporately contribute to the separation, but the hydrogen bonding effect is predominant in our HILIC separation mode.
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http://dx.doi.org/10.1016/j.chroma.2007.01.004DOI Listing
March 2007

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge.

Open Anal Chem J 2007 ;1:28-37

Center for Biochemistry and Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8014, USA.

This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans.
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http://dx.doi.org/10.2174/1874065000701010028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211269PMC
January 2007

Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography.

J Chromatogr A 2006 Nov 22;1133(1-2):119-25. Epub 2006 Aug 22.

Tokyo University of Pharmacy and Life Science, Division of Structural Biology and Analytical Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Three-phase solvent systems were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate multiple components with a wide range of hydrophobicity. The compositions of three-phase systems were optimized according to their physical parameters such as volume ratio, viscosity and specific gravity of upper (UP), middle (MP) and lower (LP) phases. The three-phase systems composed of n-hexane-methyl acetate-acetonitrile-water (4:4:3:4, v/v/v/v) was selected for HSCCC separation of a mixture of 15 standard compounds with a wide range in hydrophobicity from beta-carotene to tryptophan. The separation was initiated by filling the column with a mixture of MP and LP both as a stationary phase followed by elution with UP to separate the hydrophobic compounds. Then the mobile phase was switched to MP to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with LP. The system successfully resolved all 15 compounds in one-step operation in 70 min.
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http://dx.doi.org/10.1016/j.chroma.2006.08.004DOI Listing
November 2006

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins.

J Chromatogr B Analyt Technol Biomed Life Sci 2006 Dec 7;844(2):217-22. Epub 2006 Aug 7.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.
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http://dx.doi.org/10.1016/j.jchromb.2006.07.028DOI Listing
December 2006

Analytical separation of tea catechins and food-related polyphenols by high-speed counter-current chromatography.

J Chromatogr A 2006 Apr 18;1112(1-2):195-201. Epub 2005 Oct 18.

Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

High-speed counter-current chromatography (HSCCC) using the type-J coil planet centrifuge was applied to compositional analysis of tea catechins and separation of other food-related polyphenols. The HSCCC separation of nine different standard compounds and those from extracts of commercial tea leaves was performed with a two-phase solvent system composed of tert-butyl methyl ether-acetonitrile-0.1% aqueous trifluoroacetic acid (TFA) (2:2:3, v/v/v) by eluting the upper organic phase at a flow rate of 2 ml/min. The main compounds in the extract of non-fermented green tea were found to be monomeric catechins, their galloylated esters and caffeine. In addition to these compounds, oxidized pigments, such as hydrophobic theaflavins (TFs) and polar thearubigins (TRs) were also separated and detected from the extracts of semi-fermented oolong tea and fermented black tea. Furthermore, several food-related polyphenols, such as condensed catechin oligomers (procyanidins), phenolic acids and flavonol glycosides were clearly separated under the same HSCCC condition. These separation profiles of HSCCC provide useful information about the hydrophobic diversity of these bioactive polyphenols present in various types of teas and food products.
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http://dx.doi.org/10.1016/j.chroma.2005.09.086DOI Listing
April 2006

Alteration in endothelial function and modulation by treatment with pioglitazone in rabbit renal artery from short-term hypercholesterolemia.

Vascul Pharmacol 2005 Jun;43(1):47-55

Department of Internal Medicine, Tokyo Medical University, 6-7-1, Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan.

The present study was undertaken to investigate endothelial function and epoxyeicosatrienoic acids (EETs), which is a cytochrome P-450 monooxygenase (CYP) metabolite and one of the candidates as an endothelium-derived hyperpolarizing factor (EDHF) in the renal artery isolated from short-term hypercholesterolemic rabbits, and also to characterize the effects of pioglitazone on it. Rabbits were fed normal, 0.5% cholesterol chow, or 0.5% cholesterol chow plus 300 ppm pioglitazone for 5 weeks. The tension of isolated renal artery rings was measured isometrically. Serum lipid levels were measured and morphometric analysis was performed. EET contents in the renal artery were also determined. The cholesterol chow diet for 5 weeks increased serum lipid levels, and pioglitazone had no influence on it. In the phenylephrine precontracted renal artery, the cholesterol chow did not affect acetylcholine-induced relaxation. The N(G)-nitro-l-arginine- and indomethacin-resistant endothelium-dependent relaxation induced by acetylcholine was significantly enhanced in rabbits receiving the cholesterol chow as compared to rabbits receiving the control diet, and pioglitazone normalized it. The resistant part of acetylcholine-induced relaxation was significantly inhibited when the renal artery was treated with charybdotoxin, an inhibitor of large- and intermediate-conductance Ca(2+)-activated K(+) channels, or N,N-di-ethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525a), a nonselective CYP inhibitor, and it was significantly inhibited by sulfaphenazole, a selective CYP2C9 inhibitor in rabbits receiving only the cholesterol chow. In KCl-precontracted renal artery, the cholesterol chow inhibited acetylcholine-induced relaxation and pioglitazone normalized it. The cholesterol chow increased the production of EETs and reduced nitrate/nitrite contents in the renal artery, and pioglitazone strongly suppressed them. These results suggest that the EETs may be one of the EDHFs in the rabbit renal artery and beneficial effects of pioglitazone on alterations in endothelial function induced by cholesterol feeding are due, in part, to the protective action on the nitric oxide system and/or the suppression of increased production of EETs.
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http://dx.doi.org/10.1016/j.vph.2005.03.005DOI Listing
June 2005

Rapid and simple determination of epoxyeicosatrienoic acids in rabbit renal artery by reversed-phase HPLC with fluorescence detection.

Vascul Pharmacol 2005 Mar;42(4):163-9

Department of Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

A liquid chromatographic method with fluorescence detection coupled with a solid-phase extraction was applied to the rapid determination of epoxyeicosatrienoic acids (EETs) in the rabbit renal artery. The EETs were extracted with an acetonitrile from renal artery homogenate and concentrated by a solid-phase extraction method. The concentrated EETs were reacted directly with a 6, 7-dimethoxy-1-methyl-2 (1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ) hydrazide and separated by a reversed-phase HPLC with eluting a combination of a step-wise and a gradient of a mixture of methanol and water. The content of EETs in the renal arteries was significantly greater in the 0.5% cholesterol fed rabbits than in control rabbits. It is suggested that hyperchlesterolemia increases the production of EETs in the rabbit renal artery.
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http://dx.doi.org/10.1016/j.vph.2004.12.003DOI Listing
March 2005

Two new triterpenes from the seeds of Trichosanthes cucumeroides.

Nat Prod Res 2005 Apr;19(3):211-6

Institute of Chinese Materia Medica, China Academy of Traditional Chinese Medicine, 16 Dongzhimennei Nanxiaojie, Dongcheng, Beijing 100700, China.

Two new triterpenes named 7-oxodihydrokarounitriol (1) and 7,11-dioxodihydrokarounidiol (2), and one known triterpene, 7-oxodihydrokarounidiol (3), were isolated from the unsaponifiable matter of the seeds of Trichosanthes cucumeroides. The structures of 1 and 2 were elucidated as (3alpha, 11beta, 13alpha, 14beta, 20alpha)-3,11,29-trihydroxy-13-methyl-26-norolean-8-ene-7-one, and (3alpha,13alpha,14beta,20alpha)-3,29-dihydroxy-13-methyl-26-norolean-8-ene-7,11-dione on the basis of extensive NMR (1H, 13C, 1H-1H COSY, DEPT, HMQC, HMBC and NOESY) and MS studies.
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http://dx.doi.org/10.1080/1478641042000223790DOI Listing
April 2005

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system.

J Chromatogr B Analyt Technol Biomed Life Sci 2004 Jun;805(1):155-60

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.
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http://dx.doi.org/10.1016/j.jchromb.2004.02.039DOI Listing
June 2004

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2004 Jan;799(2):239-44

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.
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http://dx.doi.org/10.1016/j.jchromb.2003.10.039DOI Listing
January 2004

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography, partition and precipitation.

J Chromatogr B Analyt Technol Biomed Life Sci 2003 Aug;793(2):275-9

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.
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http://dx.doi.org/10.1016/s1570-0232(03)00327-1DOI Listing
August 2003

Separation of proanthocyanidins by degree of polymerization by means of size-exclusion chromatography and related techniques.

J Biochem Biophys Methods 2003 Jun;56(1-3):311-22

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The molecular masses of polyphenols in plants and food vary greatly up to the order of 10 kDa. Polymerized polyphenols are not only natural antioxidants but also strong inhibitors of numerous physiological enzymatic activities. Several useful methods for the determination and separation of these high-molecular-mass polyphenols have recently been developed. In this review, details of the methods and applications of size-exclusion chromatographic separation of polymerized polyphenols, particularly those of proanthocyanidins, are described and compared with other related chromatographic or mass spectrometric analyses.
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http://dx.doi.org/10.1016/s0165-022x(03)00068-xDOI Listing
June 2003

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2002 Sep;776(2):183-9

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.
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http://dx.doi.org/10.1016/s1570-0232(02)00348-3DOI Listing
September 2002
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