Cation-pi interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase

It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.     

Fibrous and helical calcite crystals induced by synthetic polypeptides containing o-phospho-L-serine and o-phospho-L-threonine

The modification of CaCO(3) crystal growth by synthetic L-Ser(PO(3)H(2)) and L-Thr(PO(3)H(2)) containing polypeptides is described. The amino acids Gly, L-Glu, L-Asp, L-Ser, L-Ala, and L-Lys induced rhombohedral calcite with a rough surface. Dipeptides, Xaa-L-Ser(PO(3)H(2)) (Xaa = Gly, L-Glu, L-Asp, L-Ser, L-Ala and L-Lys) induced vaterite crystals in the lower [Ca(2+)]. On the other hand, L-Ser(PO(3)H(2))-containing polypeptides formed spherical vaterite and fibrous calcite. The characteristic helical calcite was found in the presence of copoly[L-Ser(PO(3)H(2))(75)L-Asp(25)] or poly[L-Ser(PO(3)H(2))(3)-L-Asp]. Fibrous calcite, spherical vaterite, and helical calcite crystals were subjected to XRD and EDX analysis. XRD revealed the specific faces of these crystals. EDX spectra and surface analysis visualized the localization of the polypeptides and CaCO(3) components. Together with TEM and SAED data, we propose hypothetical growth mechanisms for the fibrous and helical calcite crystals.     

Effects of graft densities and chain lengths on separation of bioactive compounds by nanolayered thermoresponsive polymer brush surfaces

We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)ethyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 degrees C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by temperature-dependent elution behavior of hydrophobic steroids from the surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of solutes with the PIPAAm-grafted surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAAm at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAAm brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAAm graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAAm in dense-pack brush surfaces.     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the determination of crustacean protein in processed foods

The labeling of foods containing material derived from crustaceans such as shrimp and crab is to become mandatory in Japan because of increases in the number of allergy patients. To ensure proper labeling, 2 novel sandwich enzyme-linked immunosorbent assay (ELISA) kits for the determination of crustacean protein in processed foods, the N kit (Nissui Pharmaceutical Co., Ltd, Ibaraki, Japan) and the M kit (Maruha Nichiro Holdings, Inc., Ibaraki, Japan), have been developed. Five types of model processed foods containing 10 and/or 11.9 microg/g crustacean soluble protein were prepared for interlaboratory evaluation of the performance of these kits. The N kit displayed a relatively high level of reproducibility relative standard deviation (interlaboratory precision; 4.0-8.4% RSDR) and sufficient recovery (65-86%) for all the model processed foods. The M kit displayed sufficient reproducibility (17.6-20.5% RSDR) and a reasonably high level of recovery (82-103%). The repeatability relative standard deviation (RSDr) values regarding the detection of crustacean proteins in the 5 model foods were mostly < 5.1% RSDr for the N kit and 9.9% RSDr for the M kit. In conclusion, the results of this interlaboratory evaluation suggest that both these ELISA kits would be very useful for detecting crustacean protein in processed foods.     

Spin-crossover in cobalt(II) compounds containing terpyridine and its derivatives

In this review article we discuss the unique and novel magnetic properties for the cobalt(II) compounds with a variety of terpy derivatives including substituents at the 4-position. These are also compared with the unsubstituted terpy cobalt(II) complex. Since the first SCO cobalt(II) complex with terpy ligand was reported, this system has been widely studied. SCO cobalt(II) complexes possessing terpy or OH-terpy ligand reveal incomplete or gradual SCO behavior. The pyterpy-appended cobalt(II) complex shows SCO depending on the guest molecules involved. Cobalt(II) complexes with long-alkylated terpy ligands, [Co(Cn-terpy)₂](BF₄)₂ (n = 16, 14 and 12) have been synthesized and some were characterized by single crystal X-ray analysis. Furthermore, variable-temperature magnetic susceptibility indicated that the non-solvated compounds [Co(Cn-terpy)₂](BF₄)₂ (n = 16, 14 and 12) exhibit “reverse spin transition” phenomenon with wide thermal hysteresis around room temperature. In addition, the solvated compound [Co(C12-terpy)₂](BF₄)₂·EtOH·0.5H₂O shows “re-entrant SCO” behavior. The long alkyl chains in SCO cobalt(II) complexes can lead to novel physical properties resulting from a synergetic effect between SCO and response of the flexibility toward external stimuli.     

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.     

Thermoreversible gelation of isotactic-rich poly(N-isopropylacrylamide) in water

We report the experimentally determined phase diagram for an aqueous solution of isotactic-rich poly(N-isopropylacrylamide) (PNiPAM) composed of the sol-gel transition curve and the cloud-point curve. The meso diad content of isotactic-rich PNiPAM is 64%, and it is soluble in water at low temperatures, but undergoes a sol-to-gel transition with increasing temperature in the investigated concentration range of 1.8 wt. %-6.0 wt. %. With a further increase in temperature, the system becomes turbid. The gel formation and clouding behavior are thermally reversible. This is the first observation of thermoreversible gelation under the cloud-point temperature for an aqueous solution of PNiPAM. On the basis of the determined phase diagram, we carried out light scattering experiments to characterize the sol-gel transition behavior as a function of temperature.     

UV luminescent organic-capped ZnO quantum dots synthesized by alkoxide hydrolysis with dilute water

A novel synthesis route to organic-capped and colloidal ZnO quantum dots (QDs) has been developed. Specifically, zinc-di-t-butoxide and zinc-di-n-butoxide are hydrolyzed by very dilute water (400-600 mass ppm) in hydrophilic benzylamine and polymerized to ZnO by dehydration and/or a butanol elimination reaction. Growth of the ZnO QDs and exchange of the surface capping ligand from the hydroxyl groups and/or benzylamine to the oleylamine occur by heating the colloidal solution after addition of the oleylamine at 100-180°C. The final ZnO QDs with diameters in the range of 3-7 nm are highly dispersible in various organic solvents. The ZnO QDs exhibit the quantum size effect upon UV emission; it was controlled between 3.39 and 3.54 eV in the present study. The defect-related Vis emission decreased and the UV emission becomes dominant when zinc-di-n-butoxide with a 99.99% zinc purity is used as the starting material. The intensity of the photoluminescence UV emission is 1.5 times higher than that of the Vis emission.