Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing

A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.     

High-speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis

We developed a new system for random separation of a single microorganism, such as a living cell and a microbe, in the microfluidic device under the microscope by integrating the laser-trapping force and dielectrophoretic (DEP) force. An arbitrarily selected single microbe could be isolated in a microchannel, despite the presence of a large number of microbes in solution. Once the target microbe is trapped at the focal point of the laser, we can easily realize exclusion of excess microbes around the target by controlling the electric field, while keeping the target trapped by the laser at the focal point. To realize an efficient separation system, we proposed a new separation cell and produced it by microfabrication. Flow speed in the microchannel is adjusted and balanced to realize high-speed and high-purity extraction of the target. Some preliminary experiments are conducted to show the effectiveness. The target is trapped by the laser, transported, and is taken out from the extraction port. Total separation time is less than 20 s. Our method is extremely useful in the pure cultivation of the cell and will be a promising method for biologists in screening useful microbes.     

Tuning the Electronic Properties and Acid‐Response Behavior of N‐Heteroacene‐Based π‐Conjugated Liquids by Changing the Number of π‐Conjugated Substituents

We have designed and synthesized two room‐temperature‐fluorescent π‐conjugated liquids based on the N‐heteroacene framework ( 1 and 2 ). These two π‐conjugated liquids, which contained one and two thiophene rings, respectively, exhibited different electronic properties and rheology behaviors. Single‐crystal X‐ray analysis of dithiophene‐appended compound 4 revealed that two thiophene rings hindered the interactions of the imino N atoms with acids through the formation of interactions between the S atoms of the thiophene rings and the imino N atoms of the pyrazine group. On the other hand, monothiophene‐appended molecules 1 and 3 each contained an unhindered imino N atom on the opposite site to the thiophene ring. Upon dissolving various acids with different pK_a values in compounds 1 and 2 , these slight structural differences gave rise to marked differences in their acid‐response behaviors, thereby resulting in the emission of variously colored fluorescence in the liquid state. Furthermore, when acids with lower pK_a values was dissolved in compounds 1 and 2 , phase transition occurred from an isotropic liquid state to a self‐organized liquid‐crystalline phase.     

Temperature dependent control of the solubility of gallium nitride in supercritical ammonia using mixed mineralizer

Using a mass-loss method, we investigated the solubility change of gallium nitride (GaN) in supercritical ammonia with mixed mineralizers [ammonium chloride (NH₄Cl)?+?ammonium bromide (NH₄Br) and NH₄Cl?+?ammonium iodide (NH₄I)]. The solubilities were measured over the temperature range 450–550 °C, at 100 MPa. The solubility increased with NH₄Cl mole fraction at 450 °C and 100 MPa. The temperature dependence of the solubility curve was then measured at an equal mole ratio of the two mineralizers. The slope of the solubility–temperature relationship in the mixed mineralizer was between those of the individual mineralizers. These results show that the temperature dependence of the solubility of GaN can be controlled by the mineralizer mixture ratio. The results of the van’t Hoff plot suggest that the solubility species were unchanged over the investigated temperature range. Our approach might pave the way to realizing large, high-quality GaN crystals for future gallium-nitride electronic devices, which are increasingly on demand in the information-based age.     

Effects of a lysophosphatidylcholine and a phosphatidylcholine on the morphology of taurocholic acid-based mixed micelles as determined by small-angle X-ray scattering

We previously showed that Caco-2 cell absorption of β-carotene from taurocholic acid (TA)-based mixed micelles differed depending on the composition of the micelles. In this study, the shapes and sizes of TA-based mixed micelles, that is, mixed micelles of TA, 1-oleoyl-rac-glycerol (MG), oleic acid (OLA), and either 1-palmitoyl-sn-glycero-3-phosphocholine (MPPC; i.e., a lysophospholipid) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; i.e., a phospholipid) (60:3:1:0.75–12) were determined by using small-angle X-ray scattering (SAXS). We found that increasing the ratio of MPPC in mixed micelles of TA, MG, OLA, and MPPC was responsible for the previously observed enhanced β-carotene absorption by Caco-2 cells and changed the micelle shape from core–shell spherical to core–shell ellipsoidal. In contrast, increasing the ratio of POPC in mixed micelles of TA, MG, OLA, and POPC was responsible for the suppressed β-carotene absorption by the cells, changed the micelle shape from core–shell spherical to core–shell ellipsoidal to core–shell cylindrical, and caused a rapid increase in micelle volume. These results will be useful for understanding the mechanisms that mediate β-carotene absorption by cells and for developing technologies to improve the intestinal absorption of lipophilic components of drugs and nutrients.     

Synthesis of epigallocatechin trimer, (epigallocatechin)2-epicatechin,and (epigallocatechin)2-catechin via a Lewis acid mediated one-pot condensation and their antitumor activities in prostate cancer cells

Syntheses of epigallocatechin trimer, (epigallocatechin)₂-epicatechin and (epigallocatechin)₂-catechin were achieved. The key condensation to form the proanthocyanidin trimer derivatives was accomplished in a one-pot procedure using a dimeric epigallocatechin electrophile, which was prepared in situ by self-condensation of an epigallocatechin derivative, and an epigallocatechin, epicatechin, or catechin derivative as the nucleophile in the presence of a Lewis acid. The epigallocatechin monomer to trimer compounds containing a pyrogallol group significantly suppressed cell proliferation in PC-3 prostate cancer cells.     

Formation and Characterization of Reversed Micelles Composed of Phospholipids and Fatty Acids

The formation of reversed micellar systems composed of phosphatidylcholine (PC) and fatty acid was newly demonstrated by a significant increase in water content in the organic ethyl oleate phase when the micelles were prepared by the contact method. The solubilized water concentration in the reversed micellar organic phase reached 3 wt%. The new systems are expected to be used as highly biocompatible reversed micellar systems. The structure of the reversed micelles composed of PC and oleic acid was characterized by determining the water concentration and by small-angle X-ray scattering analysis. The reversed micelles composed of PC and oleic acid formed in ethyl oleate were spherical. The radius of gyration was between 30 and 50 Å. The size of the reversed micelles decreased with an increase in the oleic acid concentration and was independent of the PC concentration. Experimental results indicated that the structure of the reversed micellar system was determined by the oleic acid concentration. An increase in the PC concentration caused an increase in the number of reversed micelles of the same size. These reversed micellar systems are expected to be used as solubilization media in pharmaceutical and food industries because they are not toxic.     

Ab initio protein structure prediction with force field parameters derived from water-phase quantum chemical calculation

Molecular dynamics (MD) simulations are extensively used in the study of the structures and functions of proteins. Ab initio protein structure prediction is one of the most important subjects in computational biology, and many trials have been performed using MD simulation so far. Since the results of MD simulations largely depend on the force field, reliable force field parameters are indispensable for the success of MD simulation. In this work, we have modified atom charges in a standard force field on the basis of water-phase quantum chemical calculations. The modified force field turned out appropriate for ab initio protein structure prediction by the MD simulation with the generalized Born method. Detailed analysis was performed in terms of the conformational stability of amino acid residues, the stability of secondary structure of proteins, and the accuracy for prediction of protein tertiary structure, comparing the modified force field with a standard one. The energy balance between alpha-helix and beta-sheet structures was significantly improved by the modification of charge parameters.