Biomedical subjects

Yoichiro Ito

Publications and source records attributed to Yoichiro Ito.

At least 19 recordsLinked to original sources

PubMed2006

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

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.

Chromatography, Liquid↗

PubMed2006

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

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.

Countercurrent Distribution↗

PubMed2006

Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography.

High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.

Agrobacterium tumefaciens↗

PubMed2006

Preparative isolation of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb "bai zhi"Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using multidimensional high-speed counter-current chromatography.

Preparative high-speed counter-current chromatography (CCC) was successfully used for isolation and purification of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb "bai zhi"Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using multidimensional counter-current chromatography with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at volume ratios of 1:1:1:1 (v/v) and 5:5:4.5:5.5 (v/v), which had been selected by analytical high-speed counter-current chromatography (HSCCC). Using two preparative units of the CCC centrifuge, which are connected by a column switching valve, about a 300 mg amount of the crude extract was separated, yielding 19.9 mg of imperatorin, 8.6 mg of oxypeucedanin and 10.4 mg of isoimperatorin all at a high purity of over 98%.

Angelica↗

PubMed2006

Application of dual counter-current chromatography for rapid sample preparation of N-methylcarbamate pesticides in vegetable oil and citrus fruit.

Dual counter-current chromatography (dual CCC) has been successfully applied to rapid sample preparation for the simultaneous determination of residual carbaryl, fenobucarb and methomyl in vegetable oil and citrus fruit. The citrus fruit samples were extracted with n-hexane solution containing stable isotopically labeled internal standards (methomyl-d3, fenobucarb-d3 and carbaryl-d9), and applied to dual CCC using a two-phase solvent system of n-hexane-acetonitrile to purify the carbamate pesticides from aliphatic sample matrix. The coiled column was rotated at 420 rpm, the lower mobile phase was introduced through the head toward the tail, and the upper mobile phase in the opposite direction. Due to the high partition efficiency of dual CCC, the lower phase fraction collected from 2 to 5 min after injection could be subjected to flow-injection tandem mass spectrometry directly after concentration. Repetitive sample injection can be performed at high reproducibility without a risk of contamination from the compounds retained in the column.

Carbamates↗

PubMed2006

New small-scale cross-axis coil planet centrifuge. The design of the apparatus and its application to counter-current chromatographic separation of proteins with aqueous-aqueous polymer phase systems.

The cross-axis coil planet centrifuge (X-axis CPC) is useful for partitioning macromolecules with aqueous-aqueous polymer phase systems. The floor model we have built with a pair of separation columns had some shortcomings such as requirement of large space, short life of the flow tubes, and difficulty in installing columns. In order to improve the partition efficiency and the utility of counter-current chromatography (CCC), a new small-scale X-axis CPC was designed and fabricated in our laboratory. The down-sizing of the apparatus was done by reducing the scale to about 1/2 of our original model of X-1.5L type with several improvements. Performance of the apparatus was evaluated on protein separation using an aqueous-aqueous polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate with four multilayer coiled columns. A series of experiments revealed that the combination of right- and left-handed coils produced the best partition efficiencies for both lower and upper mobile phases by selecting the revolution direction. The overall results indicate that the head-tail elution mode substantially affects to the peak resolution and stationary phase retention. This new X-axis CPC would be useful for the separation of various kinds of biologically active compounds.

Centrifugation↗

PubMed2006

Evaluation of influenza vaccination in health-care workers, using rapid antigen detection test.

A study was conducted to assess the effectiveness of trivalent influenza vaccine in preventing influenza and reducing absenteeism in health-care workers of Gifu Red Cross Hospital during the 2002-2003 influenza season. Questionnaires were distributed to 370 health-care workers, and 366 were returned showing that 237 had received influenza vaccine and 129 were unvaccinated. The criterion for influenza was febrile episodes with a positive reaction on a rapid antigen detection test. Among vaccine recipients, the number of influenza infections was 3.4 per 100 subjects, compared with 8.5 per 100 subjects in nonrecipients (P = 0.034), and the number of days absent from work per 100 subjects was 9.5, compared with 15.1 per 100 subjects in nonrecipients (P = 0.0003). The prevalence of adverse reactions to the vaccine was rather low. No serious adverse reactions were recorded and no one was absent from work because of adverse reactions. It is concluded that influenza vaccine is effective in preventing influenza and reducing absenteeism in health-care workers. The results of this study support recommendations for influenza vaccination in health-care workers.

Absenteeism↗

PubMed2005

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

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.

Biflavonoids↗

PubMed2005

Isolation of a novel flavanone 6-glucoside from the flowers of Carthamus tinctorium (Honghua) by high-speed counter-current chromatography.

A novel flavanone glycoside, (2S)-4',5,6,7-tetrahydroxyflavavone 6-O-beta-D-glucopyranoside was isolated from the ethyl acetate extract of the flowers of Carthamus tinctorium by high-speed counter-current chromatography (HSCCC). Using an optimized two-phase solvent system composed of ethyl acetate-methanol-water (5:1:5, v/v), target compound (52 mg) with purity of 98.0% was obtained from 2.0 g of sample by HSCCC in seven times run. The structure of the target compound was elucidated by means of spectroscopic methods including IR, MS, 1D and 2D NMR techniques.

Carthamus↗

PubMed2005

Preparative isolation and purification of rupestonic acid from the Chinese medicinal plant Artemisia rupestris L. by high-speed counter-current chromatography.

Rupestonic acid was purified for the first time by high-speed counter-current chromatography from a dichloromethane extract of the traditional Chinese medicinal plant Artemisia rupestris L. The separation was performed in two steps with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (6:4:3.5:6.5, v/v) with 0.5% acetic acid in stationary-phase. From 200 mg of the crude extract, 27.9 mg of rupestonic acid was obtained at over 98% purity as determined by HPLC analysis, and its chemical structure was confirmed by MS, 1H and 13C nuclear magnetic resonance.

Artemisia↗

PubMed2005

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge.

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.

Acer↗

PubMed2005

Semi-industrial isolation of salicin and amygdalin from plant extracts using slow rotary counter-current chromatography.

Salicin in the bark extract of Salix alba and amygdalin in the fruit extract of Semen armeniacae were each separated by slow rotary counter-current chromatography (SRCCC). The apparatus was equipped with a 40-L column made of 17 mm i.d. convoluted Teflon tubing. A 500g amount of crude extract containing salicin at 13.5% was separated yielding 63.5 g of salicin at 95.3% purity in 20h using methyl tert-butyl ether-l-butanol (1:3) saturated by methanol-water (1:5) as a stationary phase and methanol-water (1:5) saturated by methyl tert-butyl ether-1-butanol (1:3) as a mobile phase. A 400g amount of crude extract containing amygdalin at 55.3% was isolated to yield 221.2g of amygdalin at 94.1% purity in 19h using ethyl acetate-1-butanol (1:2) saturated by water as a stationary phase and water saturated by ethyl acetate-1-butanol (1:2) as a mobile phase. The flow rate of the mobile phase was 50 ml/min. The results show that industrial SRCCC separation of salicin and amygdalin is feasible using a larger column at a higher flow rate of the mobile phase.

Amygdalin↗

PubMed2005

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.

Amino Acid Sequence↗

PubMed2005

Isolation of quercetin-3-O-L-rhamnoside from Acer truncatum Bunge by high-speed counter-current chromatography.

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of quercetin-3-O-L-rhamnoside from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase-system composed of ethyl acetate-ethanol-water at a volume ratio of 5:1:5 (v/v/v). In a single operation, 41.9mg of quercetin-3-O-L-rhamnoside was obtained from 366mg of the crude extract. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of quercetin-3-O-L-rhamnoside was over 96%. Its structure was identified by MS, 1H NMR and 13C NMR. Quercetin-3-O-L-rhamnoside was obtained from this plant for the first time.

Acer↗

PubMed2005

Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography.

This paper aims to be an aid to those chemists who are interested in utilizing high-speed counter-current chromatography (HSCCC), which is free of irreversible adsorption and offers high resolution comparable to column chromatography. It explains the selection of HSCCC conditions step by step including the selection of two-phase solvent systems, determination of partition coefficient (K) of analytes, preparation of two-phase solvent system and sample solution, selection of elution mode, flow rate, rotation speed, and on-line monitoring of the eluate. The paper covers both standard HSCCC and pH-zone-refining CCC techniques. Technical terms (italic) unfamiliar to the beginner are comprehensively explained in Glossary. Various examples of two-phase solvent systems used in HSCCC are listed in Appendices A and B. The commercial sources of HSCCC and other CCC instruments are described in detail in the study edited by Berthod [A. Berthod (Ed.), Counter-current Chromatography, Elsevier, Amsterdam, 2003].

Countercurrent Distribution↗

PubMed2005

Erectile dysfunction in patients with chronic viral liver disease: its relevance to protein malnutrition.

BACKGROUND: In patients with chronic liver disease (CLD), quality of life is generally accepted as poor, especially for physical function. However, sufficient data regarding erectile function has not been shown in patients with CLD. The international index of erectile function (IIEF) is widely used to assess erectile function, and a short form of the IIEF was recently developed (IIEF-5). Using this questionnaire, we evaluated erectile dysfunction (ED) in patients with CLD. METHODS: A total of 117 Japanese patients (64 with chronic hepatitis [CH] and 53 with liver cirrhosis [LC]) were analyzed. The etiologies were hepatitis B virus (HBV) in 21, HCV in 94, and non-B non-C in 2. The IIEF-5 and Medical Outcomes Study Short Form 36 (SF-36) were administered to the patients, and biochemical analyses for items serum albumin, prothrombin time, bilirubin, and ammonia were also performed. RESULTS: The incidence of ED was 85% in the total cohort with CLD, 78% in those with CH, and 92% in those with LC (P < 0.05 between CH and LC). ED was found in 50% of CLD patients under age 50 years, in 79% aged 50-59, and in 100% aged over 60 (P, overall <0.001). The scores for ED severity correlated with increasing grades of a modified Child-Pugh classification (P < 0.05). Simple regression analysis showed age (P < 0.01), physical function (P < 0.001), role physical (P < 0.001), and social functioning (P < 0.05) on the SF-36, and serum albumin (P < 0.001) as significant determinants of ED. Multiple regression analysis identified age (P < 0.001) and serum albumin (P < 0.001) as independent significant factors that determined ED. CONCLUSIONS: These data clearly demonstrate that liver disease is the cause of ED in patients with CLD, and serum protein status could be relevant to this condition in these patients.

Adult↗

PubMed2005

Escherichia coli O157 infection mimicking acute appendicitis: usefulness of computed tomography for differential diagnosis.

A 19-year-old man was admitted to our hospital with acute abdominal pain in the right lower quadrant. He had had mild diarrhea, of 1 day's duration, 2 days before admission. Although physical findings were consistent with a diagnosis of acute appendicitis, computed tomography findings showed marked wall thickening from the ascending colon to the cecum, findings which were similar to those in patients with hemorrhagic colitis due to Escherichia coli O157. Instead of emergency laparotomy, the patient was treated with antimicrobial agents, which led to rapid recovery. Diagnosis of intestinal infection due to E. coli O157 was established later, as serum antibody against lipopolysaccharide of E. coli O157 was positive. E. coli O157 infection should be included in the differential diagnosis of diseases that exhibit marked wall thickening of the right colon on CT in patients with acute abdominal pain in the right lower quadrant who have mild transient diarrhea.

Acute Disease↗

PubMed2004

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

Centrifugation↗