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Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model., (© 2020 by The American Society of Hematology.)

Objective With the aging of society, both osteoporosis and fatty liver disease (FLD) are becoming important issues. However, the relationship between osteoporosis and FLD remains controversial. We investigated the association between bone metabolism and FLD in a Japanese community in a cross-sectional study. Methods A total of 1,020 participants were enrolled in a health survey. FLD was diagnosed by ultrasonography. Bone metabolism was evaluated based on bone mineral density (BMD), which was assessed using dual-energy X-ray absorptiometry, and with the bone formation index (total type I procollagen N-terminal propeptide/bone-alkaline phosphatase ratio; P1NP/BAP ratio) and the bone resorption index (crosslinked N-telopeptide of type I collagen/tartrate-resistant acid phosphatase-5b ratio; NTx/TRACP-5b ratio) calculated from serum bone turnover markers. Results The BMD (percentage of the young adult mean) was the same level in both male and female participants with and without FLD. Both men and women showed an age-dependent decrease in their bone formation index and bone resorption index values. Men of ≥70 years of age and women of 60-69 years of age with FLD had significantly lower bone formation index values and higher bone resorption index values. However, similar findings were not seen in women of ≥70 years of age. Conclusion Although the BMD levels were the same, regardless of the presence or absence of FLD, elderly participants with FLD showed decreased bone formation and increased bone resorption, with sex differences. Because our results suggest that FLD in elderly individuals is detrimental for bone metabolism, and that it leads to bone loss and osteoporosis, further studies using a cohort population are warranted.

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is β'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, β'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare β'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of β'-component obtained by peptide mapping. The recombinant β'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native β'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the β'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both β'-component subunits were almost identical, and the predicted secondary structure of the β'-component showed a high content of β-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant β'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, β'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.

Miso paste (miso), a fermented soybean food, is popular in Japan and other Asian countries. However, the soybean is known to induce an allergenic response in some individuals. In the present study, we evaluated the allergenicity of various kinds of miso available in Japan. Total proteins were extracted from Amakuti-kome miso, Karakuti-kome miso, Mugi-miso and Mame-miso, and the protein profiles were analyzed. The major protein bands detected in the intact soybean extract were not present in any of the miso samples, which instead showed various low molecular weight protein bands of approximately 10-25 kDa. The existence levels of six major soybean allergens were determined by Western blotting using specific antibodies. We found that the allergen levels varied among miso and allergen types; however, allergen levels were consistently lower in miso than in the soybean extract. We obtained similar results for IgE-ELISA experiments using serum IgE from soybean allergy patients. Taken together, these results indicate that compared to soybean extract, various types of miso contain small quantities of intact soybean allergens. Additionally, several lines of evidence indicated that the allergen levels were exceptionally low in the dark-colored Karakuti-kome miso and Mame-miso, which are produced with relatively long fermentation periods, suggesting that the duration of fermentation might be a key factor in the hypoallergenicity of miso.

Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

Background: MUC-1 is synthesized as a single polypeptide that then undergoes proteolytic cleavage, and is associated with the epidermal growth factor receptor tyrosine kinases. In malignancies, MUC-1 may function as an anti-adhesion molecule, but can also promote adhesion and presumably metastasis., Materials and Methods: Expression of MUC-1, -2, -4 and -5AC was evaluated on tissue microarrays of gastric carcinomas (n = 237) and adjacent non-cancerous mucosa specimens (n = 89) by immunohistochemistry and compared with clinicopathological parameters and survival time of the patients., Results: MUC-1 was found to be highly expressed in gastric carcinomas in comparison with noncancerous mucosa (p < 0.05) and positively correlated with depth of invasion, lymphatic and venous invasion, lymph node metastasis, UICC staging and MUC-4 expression (p < 0.05), but not with age, tumor size, MUC-2 or MUC-5AC expression (p > 0.05). Intestinal-type carcinomas showed more MUC-1 expression than their diffuse-type counterparts (p < 0.05). Kaplan-Meier analysis indicated that the cumulative survival rate of patients with no MUC-1 expression was significantly higher than those with weak, moderate or strong expression in gastric carcinomas (p < 0.05), but no difference was observed when tumors were stratified according to the depth of invasion (p > 0.05). Cox's analysis showed three independent prognostic factors, depth of invasion, lymphatic invasion and venous invasion, to affect the relationship between MUC-1 expression and prognosis., Conclusion: Up-regulation of MUC-1 expression may be involved in pathogenesis, invasion, metastasis and differentiation of gastric carcinoma. Altered expression might therefore be employed as an indicator of pathobiological behavior of gastric carcinoma. MUC-1 expression was found to be a prognostic factor for gastric carcinoma patients, albeit not independent of parameters of invasion.