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Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4–S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.

We developed a colorimetric analytical method for favipiravir (FPV), a promising treatment for COVID-19. FPV forms yellow complexes with ferrihydrite (Fh) by a ligand substitution reaction with the iron (III) hydroxyl surface groups in Fh. Fh-coated microbeads were packed into a capillary tube with an inner diameter of 1 mm. FPV-spiked serum after pretreatment was drawn into the capillary packed with Fh-coated microbeads. The intensities of the yellow-color complexes on the microbeads were transformed into red-green-blue (RGB) pixels using Image-J software 1.8, and the difference between green and blue was used as the analytical signal. The signal increased with increasing FPV concentration. The proposed method showed good linearity (R

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.

We developed a handheld fluorescence detection system for capillary-based enzyme-linked immunosorbent assay (ELISA). The detection system implements both a long-pass filter and perpendicular optical arrangement, i.e., a power LED and a palm-sized spectrometer, to minimize background signals from the excitation light and optical scattering. The lower detection limit for resorufin was 0.13 μM. The detection system was applied to the quantification of C-reactive protein (CRP) in human serum with a capillary-based ELISA. The lower detection limit for CRP was 31 ng/ml, and the observed CRP levels in human serum were comparable to those obtained with a conventional ELISA system.

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

We report that the channel activities of gramicidin A in a supported lipid bilayer (SLB) were modulated by membrane fusion with exosomes. The mechanism of the modulation was an increase in the number of exosomes inserting into the SLB membrane, rather than enhancements of the single channel activity of gramicidin A. The modulation of apparent channel activities was applicable to the exosome fusion assay. This assay revealed that the membrane fusion of HEK-293 and MCF-7 exosomes was enhanced at pH 6.0, and the initial rates of membrane fusion for MCF-7 exosomes were higher than those for HEK-293 cells.

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.

A chromanone glycoside, 5-β- d -glucopyranosyloxy-7-hydroxy-2-isopropylchromanone, and two acylphloroglucinol glycosides, 2-(3-methylbutyryl)phloroglucinol-4,6-di-C-β- d -glucopyranoside and 2-isobutyrylphloroglucinol-1,5-di-O-β- d -glucopyranoside, along with 15 known compounds were isolated from the bracts of hops (Humulus lupulus L.). Their structures were elucidated based on spectroscopic data, including TOFMS and 1D/2D NMR.

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.

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.

Aqueous–aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, Mr:1000–8000) and dextran (Mr: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.

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 10 mM 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.

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.

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.

A new method was developed for the fractionation of procyanidin oligomers according to their degree of polymerization. Monomeric flavan-3-ols and low molecular mass procyanidins were selectively extracted from the lyophilized powder of apple condensed tannins (ACTs) by methyl acetate extraction. Sequentially, the separation of each oligomer from dimer to pentamer in this extract was carried out by normal-phase high-performance liquid chromatography using a silica-beads packed column. The best separation was achieved with a mobile phase system containing hexane; (1) hexane-methanol-ethyl acetate, (2) hexane-acetone. These sequential treatments can be easily adapted to large-scale fractionation.