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To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared single amino acid substituted recombinant BLG mutants (BLG/P126A, BLG/V128D and BLG/D129A) in the methylotrophic yeast Pichia Pastris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae . Isoelectric points of single amino acid substituted BLGs were lower than that of native BLG. CD spectra indicated that the secondary structure of BLG had maintained native structure in single amino acid substituted BLGs. Fluorescence studies indicated that the conformation around Trp had not changed in single amino acid substituted BLGs. Anti-BLG antibody response was evaluated after immunization to C57BL/6 mice. Antibody response was reduced after immunization with BLG/P126A, BLG/V128D and BLG/D129A. And novel immunogenicity was not observed in the experiments. T cell proliferative response was evaluated in C57BL/6 mice, and it was clarified that BLG mutants also showed low response. Methods employed in this study was considered to be very effective to reduce immunogenicity of BLG., Competing Interests: Conflict of interestThe authors have no conflicts of interest to declare., (© The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.

To investigate the effects of processing Chinese quince fruit on the denaturation of phenolics and their food functions, fruit phenolic extracts were heated together with organic acid for up to 12 h. Chinese quince phenolic (mostly procyanidins) solution subjected to heat treatment changed from almost colorless, pale yellow, to a reddish color. Before heat treatment, the absorption spectra of polymeric procyanidins were observed only around 280 nm; after heat treatment, absorption occurred between 400 and 600 nm, which is related to the reddish color appearance. Thioacidolysis of denatured reddish phenolics showed that (-)-epicatechin subunits decreased during heat treatment and, in contrast, cyanidin increased. In addition, novel substances that could not be degraded by thioacidolysis were formed. Meanwhile, antioxidant activities, assessed by linoleic acid peroxidation, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Folin-Ciocalteu, and FRAP methods, increased during heat treatment. The antiinfluenza viral activity of denatured reddish phenolics was inferior to that of intact fruit phenolics; however, they retained moderate activity. These results indicate that red coloration of fruit products of Chinese quince was mainly due to the spectral (i.e. structural) changes of procyanidins accompanied with formation of cyanidin. Increasing the length of heat treatment increased the antioxidant capacity of phenolics, and the resultant reddish phenolics retained moderate antiinfluenza viral activity.

To evaluate the health benefits of Chinese quince and quince phenolics, their antioxidant properties and antiulcerative activity were investigated in comparison with apple phenolics as a reference. The strength of antioxidant activity and DPPH radical scavenging activity of these fruit phenolics varied according to different in vitro evaluation systems, whereas the antioxidative property of rat blood increased in all rats orally administered phenolics. Ferulic acid and isoferulic acid were detected as major metabolites in rats given apple phenolics, quince phenolics, and 5-caffeoylquinic acid standard. (-)-Epicatechin and its 3'-O-methyl ether could be detected in rats administered apple phenolics and (-)-epicatechin standard. In the ethanol-induced gastric ulcer, pre-administration of Chinese quince and quince phenolics suppressed the occurrence of gastric lesions in rats, whereas apple phenolics seemed to promote ulceration. The trend of myeloperoxidase activity was similar to that of the ulcer index. The results showed that Chinese quince and quince phenolics might have health benefits by acting both in blood vessels and on the gastrointestinal tract.

To evaluate the phenolic extracts of Chinese quince, quince, and apple fruits, their phenolic profiles, antioxidant properties, and anti-influenza viral activities were investigated. Chinese quince had the largest amount of phenolics consisting mainly of high polymeric procyanidins. Quince had considerable amounts of hydroxycinnamic derivatives mainly composed of 3-caffeoylquinic acid and 5-caffeoylquinic acid and polymeric procyanidins. Apple (cv. Fuji) had the lowest amount of phenolics, mainly 5-caffeoylquinic acid and monomeric and oligomeric procyanidins. The antioxidant functions of Chinese quince and quince phenolic extracts were superior to that of chlorogenic acid standard or ascorbic acid evaluated in both the linoleic acid peroxidation system and the DPPH radical scavenging system. However, those extracts were less effective than apple phenolics or (-)-epicatechin in linoleic acid peroxidation system. On the other hand, Chinese quince phenolics showed the strongest anti-influenza viral activity on the hemagglutination inhibition test.

Thrombomodulin is a glycoprotein located on the surface membrane of vascular endothelial cells. Recently the presence of thrombomodulin antigens was detected in circulating blood plasma. The thrombomodulin antigens are thought to have been released from injured endothelial cells, and thus plasma thrombomodulin has been attracting attention as a new molecular marker indicating injuries to vascular endothelial cells. We investigated plasma thrombomodulin levels in relation to the severity of diabetic retinopathy, and found that they increased as retinopathy progressed. The Mann-Whitney U test revealed significant differences between no retinopathy and proliferative retinopathy (p = 0.002), between simple retinopathy and proliferative retinopathy (p = 0.01), and between preproliferative retinopathy and proliferative retinopathy (p = 0.03). However, ocular vascular injuries may correlate with systemic vascular injuries. Therefore, the progress of retinopathy may be predicted by monitoring plasma thrombomodulin levels in selected patients.