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Complicated phase transitions were observed in a single-component 1-decyl-3-methylimidazolium nitrate ([C 10 mim][NO 3 ]) ionic liquid (IL) using Raman spectroscopy and synchrotron small- and wide-angle X-ray scattering (SWAXS). Time-resolved synchrotron SWAXS could distinguish the phase transitions depending upon the cooling rate. Low- Q peaks representing a few kinds of layered structures were decomposed. Multiphase coexistence was observed in [C 10 mim][NO 3 ] at specific cooling rates (8-9 K/min). Ionic liquid crystals (ILCs), hybrid-layered crystals, and hexagonal close-packed structures coexisted simultaneously. At the cooling rate region, the reentrant phase transition of the ILC phase upon heating was observed.

Trypsin inhibitors (TIs) have various nutritional effects. However, a detailed mechanism for their effects, especially on the gene expression patterns in various tissues, remains unknown. Here, we used transcriptome techniques and gene ontology (GO) analysis to examine the effects of squid TI (sqTI), a biochemically stable peptide, on diabetic Goto-Kakizaki rats after feeding for 10 weeks. We demonstrated that downregulation of SREBP1c in the liver via duodenal/pancreatic hormones suppresses the blood cholesterol level. Consistently, in GO analysis, the term "cholesterol biosynthetic process" was enriched among downregulated genes. No hypoglycemic or insulinotropic effects were observed, in contrast to the results from our previous studies (single stimulation with the same dose of TI), which can be partly ascribed to the inactive responses of the duodenum and pancreas in this condition.

To investigate the effects of oral administration of a trypsin inhibitor (TI), normal Wistar rats were fed a TI derived from squid (Todarodes pacificus) for 10 weeks and gene expression profiles in the duodenum, pancreas, liver, and muscle were then analyzed using DNA microarrays. Although no significant changes could be observed in growth, food intake, tissue weight, or blood tests among the tissues tested, the duodenum showed the most remarkable changes in the global gene expression profile. Significant up-regulation of mRNAs encoding gastrin, gastrokine, cholecystokinin and somatostatin in the duodenum was validated by qPCR analysis. In gene ontology (GO) analysis of the up-regulated differentially expressed genes (DEGs), GO terms related to keratinization and innate mucosal defense were enriched (p < 0.001) in the category of biological processes in addition to assumable terms such as regulation of secretion and response to nutrients, vesicle-mediated transport, and so forth. In the same analysis, calcium ion binding was listed at the deepest hierarchy in the category of molecular function. These results indicate that the duodenum responds to TI treatment by a wider range of physiological processes than previously assumed such as keratinocyte differentiation and innate mucosal defense, in which calcium plays a crucial role.

Trypsin from the pyloric ceca of pectoral rattail (Coryphaenoides pectoralis) was purified and characterized. Purification was carried out by ammonium sulfate precipitation, followed by column chromatographies on Sephacryl S-200, DEAE-cellulose and Sephadex G-50. The enzyme was purified 89-fold with a yield of 2.2%. Purified trypsin had an apparent molecular weight of 24 kDa when analyzed using SDS-PAGE and size exclusion chromatography. Optimal profiles of pH and temperature of the enzyme were 8.5 and 45 degrees C, respectively, using N(alpha)-p-tosyl-l-arginine methyl ester hydrochloride as a substrate. It was stable in a wide pH range of 6-11 but unstable at a temperature greater than 40 degrees C. Trypsin was stabilized by calcium ion. The activity of purified trypsin was effectively inhibited by soybean trypsin inhibitor and TLCK and was partially inhibited by EDTA. Activity continuously decreased with increasing NaCl concentration (0-30%). The kinetic trypsin constants K(m) and K(cat) were 0.15 mM and 210 s(-1), respectively, while the catalytic efficiency (K(cat)/K(m)) was 1400 s(-1) mM(-1). The N-terminal amino acid sequence of trypsin was determined to be 12 residues (IVGGYECQEHSQ), and the sequence showed high homology to other fish trypsins.

Yolk protein extracts were prepared from four kinds of salmonid fish roes, and the proteins that reacted with IgE were screened by immunoblotting using sera from 20 patients allergic to chum salmon roe. IgE cross-reactivities among the salmonid yolk proteins were also investigated by competitive ELISA. The results were as follows: (1) The major protein components in salmonid roes were lipovitellin and beta'-component, which are subfragments of vitellogenin. (2) Most sera from the patients showed IgE reactivity to beta'-component in all yolk protein extracts, and some of them also reacted to lipovitellin heavy chain or its light chain. (3) Salmonid beta'-component showed high similarity (>90%) in the N-terminal amino acid sequence. (4) All of the salmonid yolk protein extracts inhibited the IgE reaction between patient sera and the chum salmon beta'-component. These findings indicate that the beta'-component in salmonid roe is a common major allergen with strong IgE cross-reactivity.

Trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda) was purified and characterized with respect to its purity; molecular weight; sensitivity to temperature, pH, and inhibition; and N-terminal sequence. The purified trypsin had a molecular weight of 29 kDa as per sodium dodecyl sulfate polyacrylamide gel electrophoresis, and optimal activity was observed at pH 9 and 65 degrees C with BAPNA as a substrate. The enzyme was stable to heat treatment up to 50 degrees C and within the pH range of 7-12. It was stabilized by calcium ions, but its activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and phenyl methyl sulfonyl fluoride. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration (0-30%). The N-terminal 20 amino acid residues of Atlantic bonito trypsin were determined as IVGGYECQAHSQPWQPVLNS and were homologous with other trypsins.

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.

Alginate oligosaccharide (AO) was conjugated with carp myofibrillar protein (Mf) by using the controlled Maillard reaction, and the change in the solubility of Mf in low ionic strength media as affected by the glycosylation was investigated. AO was prepared by degrading sodium alginate using alginate lyase, which was purified from the culture supernatant of Pseudoalteromonas elyakovii. When a lyophilized Mf and AO mixture was incubated at 40 degrees C and 65% relative humidity, the conjugation of AO was confirmed at myosin heavy chain, actin, and tropomyosin. When >30 microg/mg of AO was conjugated to Mf, the protein solubility in a low ionic strength medium was greatly improved without significant loss of available lysine. These results indicate that the conjugation with AO is a superior manner for improving the water solubility of Mf in view of its nutritional aspect.