We have shown that methanol extract of mango leaves (MLE) has strong anti-inflammatory effects in an experimental system using LPS-activated macrophage-like Raw264.7 cells. Therefore, we attempted to elucidate the molecular mechanism of the anti-inflammatory effect of MLE by quantitative proteomic analysis. The results showed a decrease in key factors of the NFkB pathway (p38 MAPK and NFkB p65) and an increase in autophagy-related factor p62, which may be related to the anti-inflammatory effect of MLE.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Background: Psoriasis is a refractory inflammatory disease, however, its pathophysiology is still not fully understood. Objective: We tried to identify novel serum peptides associated with the pathophysiology of psoriasis. Methods: Serum peptides from 24 patients with psoriasis vulgaris (PV), 10 patients with psoriatic arthritis (PsA),14 patients with atopic dermatitis (AD), and 23 healthy control (HC) subjects were analyzed by mass spectrometry. The effects of some peptides on the secretion of humoral factors from dermal cells were investigated by cytokine arrays and ELISAs. Results: A total of 93 peptides were detected. 24, 20, 23, and 2 peptides showed at least 1.2-fold difference in ion intensity between the psoriasis (PV + PsA) and HC groups, between the PV + PsA and AD groups, between the PV and PsA groups, and between patients with severe-to-moderate PV (n = 6) and those with mild PV (n = 18), respectively (p < 0.05). 13 out of 27 peptides that showed at least 1.5-fold ion intensity difference in the abovementioned 4 comparisons were identified. The parent proteins of the identified peptides included a coagulation factor,, proteins involved in the maintenance of skin, and a protein relating to cytoskeleton. We focused on 2 peptides that were increased in the PV + PsA group: a fibrinogen a chain-derived peptide (1462 m/z), the unmodified form of which was fibrinopeptide A-desalanine (FPAdA), and a filaggrin (FLG)-derived peptide (1977 m/z), a modified form of FLG(2099-2118) (Q(2099)pE, Q(2115)E; FLG-pEE). FPAdA stimulation increased the secretion of GRO alpha from dermal microvascular endothelial cells (dMVECs) and decreased the secretion of lipocalin-2 from keratinocytes in comparison to FPAdA-resequenced peptide stimulation (GRO alpha, 280.9 +/- 7.3 pg/mL vs. 229.6 +/- 5.0 pg/mL, p < 0.001; lipocalin-2, 273 +/- 13 pg/mL vs. 350 +/- 10 pg/mL, p < 0.01). Interestingly, FLG-pEE stimulation decreased the secretion of GRO alpha, IL-8, and MCP-1 from dMVECs in comparison to FLG-derived control peptide stimulation (GRO alpha, 844.3 +/- 47.5 pg/mL vs. 1038.5 +/- 96.9 pg/mL, p < 0.05; IL-8, 2240.1 +/- 172.6 pg/mL vs. 3221.8 +/- 523.7 pg/mL, p < 0.05; MCP-1, 4057.8 +/- 157.2 pg/mL vs. 4619.1 +/- 213.4 pg/mL, p < 0.05). Conclusions: The results suggested that some serum peptides are involved in the pathophysiology of psoriasis, regulating the secretion of inflammatory chemokines and an antimicrobial protein. The modulation of serum peptides may be a potential therapeutic strategy for psoriasis. (C) 2017 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Objective Post-translational modifications (PTMs) are often critical for the function of proteins as well as antigenicity of proteins. We here tried to elucidate alteration of PTMs in Rheumatoid arthritis (RA), focusing on acetylation. We applied acetyl-proteomics to peripheral blood mononuclear cells (PBMCs) to elucidate PTM difference between patients with RA and healthy donors. Methods Proteins, extracted from peripheral blood mononuclear cells (PBMCs) of 7 RA patients and 7 healthy donors, were separated by 2-dimansional electrophoresis. Acetylation ratios of each protein spot were estimated by the combination of Sypro Ruby staining and anti-acetylated lysine antibodies. Proteins highly acetylated in the RA group were identified by mass spectrometry. Focusing on alpha-enolase (ENO1), one of the identified proteins, involvement of histone deacetylases (HDACs) in the high acetylation was investigated. Furthermore, the effects of acetylation on the activity of ENO1 were investigated. Results In PBMCs from the patients with RA, 29 acetylated protein spots were detected. One of highly acetylated proteins in the RA patients was identified as ENO1. The acetylation of ENO1 was found to be regulated in part by HDAC1. The enzymatic activity of ENO1 was up-regulated by acetylation. Conclusion Highly acetylated ENO1 may play roles in the pathophysiology of RA through the maintenance of activated lymphocytes by increasing glycolysis-derived energy supply.

Early assessment of carcass traits and meat quality characteristics during fattening is expected to improve the efficiency of beef cattle production. Yet, there have been few reports on developing an assessment index for feeding condition of Japanese Black cattle. In this study, to identify protein biomarkers for predicting carcass traits and meat quality characteristics of Japanese Black cattle by proteomics, we first compared protein expression profiles of perirenal white adipose tissue between low and high merit steers in five carcass traits (carcass weight, rib eye area, rib thickness, subcutaneous fat thickness, and beef marbling standard [BMS] number). We then investigated the involvement of the lower- or higher-expressed proteins in metabolic pathways and evaluated the expression of vimentin proteins by western blot analysis in individual steers. We identified 45 proteins which were significantly expressed in low or high merit groups of five carcass traits. Coordinate involvement of some identified proteins in glycolysis/gluconeogenesis and citrate cycle pathways was found in high merit groups of carcass weight, subcutaneous fat thickness and BMS number. The expression of vimentin protein, which was significantly expressed in the high BMS number group, was confirmed in individual steers. Taken together, these results suggest the possibility of these identified proteins as protein biomarker candidates for predicting carcass traits and meat quality characteristics during fattening of Japanese Black cattle.

Objective: To find a blood biomarker and disease-related peptides in Alzheimer's disease (AD), we comprehensively detected serum peptides. Methods: Ion intensity of serum peptides from 62 AD patients and 82 control subjects was measured by mass spectrometry. Results: A total of 157 peptides were detected from 30 AD patients and 30 healthy control (HC) subjects. Sixty out of the 157 peptide profiles discriminated between the AD and HC groups. Sixteen out of the 60 peptides were identified, 10 out of which were fragments of a fibrinogen alpha chain (FIBA). Among the 10 peptides, four and six peptides were derived from fibrinopeptide A (FPA, A alpha 1-16) and the C-terminal region of the alpha C-domain (alpha CDC, A alpha 557-610), respectively. The profile of 10 FIBA-derived peptides combined with age discriminated between the two groups with an area under the receiver operating characteristic curve (AUROC) of 0.940. Validation of this model using a testing set of 32 AD patients and 19 HC subjects showed an AUROC of 0.717, sensitivity of 65.6%, and specificity of 73.7% by a cutoff value of 0.56420. Another value, 0.04029, showed sensitivity of 96.9%, suggesting that subjects with values less than 0.04029 rarely possess AD. FPA and alpha CDC showed increased ion intensity in the AD group compared with the HC group (p < 0.05). Conclusions: The profile of 10 FIBA-derived peptides combined with age would be a candidate biomarker for AD, which facilitates screening of the disease. The significant release of FPA and alpha CDC may be involved in the aberrant coagulation that leads to vascular damage in AD. Copyright (C) 2013 John Wiley & Sons, Ltd.

Objective. To investigate the pathophysiology of Behcet's disease (BD) and find biomarkers for the disease, we analysed protein profiles of peripheral blood mononuclear cells (PBMCs). Methods. Proteins, extracted from PBMCs, were comprehensively analysed in 16 patients with BD, 16 patients with rheumatoid arthritis (RA), 12 patients with Crohn's disease (CD), and 16 healthy control subjects (HC) by 2-dimensional differential gel electrophoresis (2D-DIGE). Differently expressed proteins were identified by mass spectrometry. Results. 563 protein spots were detected. We completely discriminated between the BD and HC groups, between the BD and RA groups, and between the BD and CD groups by multivariate analysis of intensity of 23, 35, and I spots, respectively. The spots contributing to the differences included proteins related to cytoskeleton, transcription! translation, T cell activation, bone turnover, regulating apoptosis, and microbial infection. Intensity of 3 spots (tyrosine-protein phosphatase non-receptor type 4, threonine synthase-like 2, and beta-actin) provided area under the receiver operating characteristic curves (AUROC) of 0.889 for discrimination between the BD group and the non-BD groups. Informatively, intensity of the above I spot completely discriminated the CD group from the other groups (AUROC 1.000). This spot, identified as beta-actin, had different pI from the above beta-actin-spot probably due to different post-translational modification. Conclusion. PBMC protein profiles, especially the profile of the 3 spots, would be candidate biomarkers for BD. The latter beta-actin subtype would be useful for discriminating inflammatory bowel diseases from BD and other diseases. The identified proteins may play important roles in the pathophysiology of BD.

Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p< . 0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various p. I values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. Biological significance: In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA. © 2013 Elsevier B.V.

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4-7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.

Aim: Biomarkers predicting sustained virological response (SVR) to pegylated interferon-α plus ribavirin (PEG IFN-α/RBV) were investigated. Methods: Peptides in pretreatment sera from 107 patients with hepatitis C virus (HCV) genotype 1 were comprehensively analyzed by mass spectrometry. Ion intensity of the peptides was used to generate discriminant models between the responders who achieved SVR (R) and the non-responders (NR) to PEG IFN-α/RBV. Results: In total, 107 peptides were detected in a training set (n = 23). A discriminant model using a peptide, complement 3f des-arginine (C3f-dR), showed sensitivity of 35% and specificity of 94% for SVR prediction in a testing set (n = 68). In all the R and NR (n = 96), an area under the receiver-operator curve (AUROC) of 0.64 in the C3f-dR model was increased to 0.78 by addition of platelet (PLT) counts (C3f-dR/PLT model). Another model using the 107 peptides (AUROC, 0.77) also showed higher AUROC (0.79) by addition of hemoglobin (Hb), body mass index (BMI) and age (107P/Hb/BMI/Age model). The sensitivity and specificity of the C3f-dR/PLT model were 59% and 88%, and those of the 107P/Hb/BMI/Age model were 70% and 92%, respectively. The C3f-dR/PLT model showed high AUROC (0.82), similar to that of interleukin-28B rs8099917 genotype analysis (0.86) in the 45 tested patients. Prediction by the combination of the C3f-dR/PLT model, the 107P/Hb/BMI/Age model and the rs8099917 genotype analysis was accurate in 44 out of the 45 patients (AUROC, 0.95). Conclusion: Serum peptides, especially C3f-dR, would be useful predictors for SVR to PEG IFN-α/RBV. The complements may be involved in the HCV elimination. © 2012 The Japan Society of Hepatology.

【目的】母性遺伝子由来転写産物やタンパク質は，卵胞発育，受精及び胚発生に重要な役割を果たしている。これまでの研究から，母性遺伝子の機能解析において糖タンパク質の一つであるZp3のプロモーターが用いられている（Millar et al., 1991; Linang et al., 1997）。しかし，原始卵胞ではこのプロモーターの転写活性が認められず，また卵母細胞で発現する他のプロモーターについても情報は少ない。我々は，卵母細胞における母性遺伝子の機能解析を行うためのツールとしてHistone H1foo（H1oo）のプロモーター領域を単離し（Tsunemoto et al., 2008），下流にホタルルシフェラーゼ遺伝子を結合した導入遺伝子を組み込んだトランスジェニックマウスを3系統（H14・H16・H19系統）作出した。本実験では，これらのトランスジェニックマウスにおける導入遺伝子の発現を明らかにすることを目的にトランスジェニックマウス由来卵母細胞及び初期胚，各組織におけるルシフェラーゼの発現を検討した。【方法】導入遺伝子が導入された雌マウスに過排卵処置を行い体外受精を施した。培養後，各発生段階でシングルフォトンイメージング装置を用いてルシフェラーゼ活性を測定した。また，各組織をタンパク質抽出し，ルミノメーターを用いて発光量を測定した。【結果及び考察】H16系統での卵母細胞及び初期胚で導入遺伝子の発現が認められ，H1ooの転写制御下でマーカー遺伝子であるホタルルシフェラーゼが発現していることが明らかになった。

Anti-ribonucleoprotein (anti-RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1-68k), a major antigen of anti-RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1-68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1-68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1-68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti-RNP antibodies.

Proteomic profiling on Ecklonia cava Kjellman grown under various seawater temperatures was conducted to search for biomarkers that were useful to evaluate the health of the colonies and formulate actions for the maintenance of marine forests. In the cultivated strains, protein expression was not significantly changed when the cultivation temperature was lowered from 15A degrees C (control) to 10A degrees C. On the contrary, it was markedly changed, i.e., photosynthesis-related proteins were up-regulated and metabolic enzymes were down-regulated, when the temperature was heightened to 20A degrees C. With the cultivation at 30A degrees C, 25 spots within 27 spots expressed at this temperature peculiarly could be identified and classified into ten proteins. Of the distinctive 27 spots at 30A degrees C, 20 spots were detected in the wild strains cultured at the same temperature for a brief time. It is presumed that the proteins including vanadium-dependent bromoperoxidase are heat stress-induced proteins.

Objective. Microscopic polyangiitis (MPA) is necrotizing vasculitis of unknown etiology. We analyzed the serum peptide profile of MPA to find a biomarker for this disease. Methods. Serum peptides from 33 patients with MPA, 7 with granulomatosis with polyangiitis (Wegener's), 7 with Churg-Strauss syndrome, 6 with giant cell arteritis, and 25 with systemic lupus erythematosus (SLE) were comprehensively analyzed by mass spectrometry. Peptide function on human microvascular endothelial cells (HMVECs) was examined by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Results. A total of 102 serum peptides were detected from the 78 patients. One of the peptides, peptide 1,523, showed significantly higher ion intensity in MPA (mean +/- SD 46.8 +/- 39.3 arbitrary units [AU]) than in the other systemic vasculitides (14.1 +/- 12.2 AU) (P < 0.05) or in SLE (17.0 +/- 12.1 AU) (P < 0.05). In MPA, peptide 1,523 showed significantly higher ion intensity before treatment than 1 week (P < 0.05) and 6 weeks (P < 0.05) after the initiation of treatment. Peptide 1,523 was identified as 13 C-terminal amino acid residues of apolipoprotein A-I (Apo A-I) and was designated "AC13." Validation of AC13 ion intensity using another MPA cohort (n = 14) similarly showed significantly higher ion intensity (90.1 +/- 167.9 AU) compared to 14 patients with rheumatoid arthritis (8.6 +/- 5.4 AU) (P < 0.01) and 14 healthy subjects (11.8 +/- 6.1 AU) (P < 0.01). Serum concentrations of Apo A-I and hig-density lipoprotein cholesterol were down-regulated in MPA before treatment and returned to their normal ranges 6 weeks after the initiation of treatment (both P < 0.01). Stimulation of HMVECs with AC13 significantly up-regulated secretion of interleukin-6 (IL-6) (P < 0.05) and IL-8 (P < 0.01). Conclusion. AC13, a candidate biomarker for MPA, may be useful for monitoring disease activity and may exacerbate vascular inflammation through upregulation of proinflammatory cytokines.

Aim. The mechanisms underlying the formation of abdominal aortic aneurysms have yet to be fully clarified. To identify key proteins generally involved in aneurysmal formation, proteomic profiles were compared between aneurysmal and non-aneurysmal regions of aortic walls from patients with abdominal aortic aneurysm. Methods. Aortic wall specimens were obtained from three patients with abdominal aortic aneurysm. Protein profiles of aortic wall samples including vascular media and adventitia were compared between aneurysmal and non-aneurysmal regions in each patient using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Protein spots expressed differently between the two regions were identified by tandem mass spectrometry and verified by immunohistochemical investigations. Results. Image analysis of 2D-DIGE gels revealed 22 proteins spots expressed differently between aneurysmal and non-aneurysmal regions in all three patients. Among these, five protein spots that were up-regulated in the AA regions were successfully identified as complement component C4, fragments of the fibrinogen alpha or beta subunits, and actin. immunohistochemical studies showed massive deposition of fibrin/fibrinogen or its fragments in the media, and complement C1q component, the molecule starting the classical complement pathway, in all three layers of the aneurysmal region. Conclusion. Our proteomic and subsequent immunohistochemical studies revealed significant fibrinogenesis and fibrinolysis in the media, and activation of the classical complement pathway in all three layers of the aneurysmal region. These data promote understanding of mechanisms behind the formation of abdominal aortic aneurysms.

Objective To identify novel proteins involved in the pathogenesis of rheumatoid arthritis (RA) and to characterise the identified proteins based on pathogenic and therapeutic aspects. Methods The authors applied differential phosphoproteomic analysis to articular synoviocytes between RA and osteoarthritis (OA) to identify proteins differently phosphorylated between RA and OA. Focusing on annexin VII (Anx7), one of the highly phosphorylated proteins in RA, the authors prepared Anx7-transgenic C57BL/6 (Anx7-Tg-B6) mice to evaluate their susceptibility to collagen-induced arthritis (CIA). In addition, the authors examined the effect of anti-Anx7 antibodies (Abs) on CIA and serum levels of cytokines in wild-type DBA/1J mice, which are known to be susceptible to CIA, and in Anx7-Tg-B6 mice. In vitro, the authors examined the effect of the Anx7 knockdown by small interfering RNA on the secretion of cytokines in rheumatoid synoviocytes and the human synovial sarcoma cell line SW982. Results The Anx7 transgene altered the CIA-resistant B6 mice to CIA-susceptible ones. The Abs treatment suppressed CIA even in the wild-type DBA/1J mice. The serum levels of cytokines including interleukin 6 (IL-6) and TNF alpha were not altered by the Abs treatment in vivo. On the other hand, the knockdown of Anx7 by small interfering RNA caused downregulation of IL-8 secretion in vitro. Conclusions These results indicate that Anx7 participates in the pathogenesis of RA partly through the secretion of IL-8. The study data have demonstrated the pathogenic roles and therapeutic significance of Anx7 in RA for the first time.

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.

Renal biopsy samples are important not only for the diagnosis of glomerulonephritis, but also for the investigation of its pathogenesis. However, it remains difficult to biochemically analyze proteins extracted solely from the glomeruli of needle biopsy samples, since the samples contain various components like renal tubules and connective tissue. Even a recent micro-dissection method, recovering the glomeruli in the sliced sections of the biopsy samples, has not fully solved the difficulty because the amount of obtainable proteins by this method is not usually enough for protein analysis. To overcome this problem, we established a simple but reliable method to isolate whole glomeruli from needle biopsy samples. By this method, termed 'micro-sieving', we were able to isolate on average more than 50 glomeruli from a single needle biopsy sample in an hour. The amount of the extracted glomerular proteins was on average 23 mu g per biopsy sample. As a representative use of this method, we were able to obtain a glomerular protein profile by fluorescent 2-dimensional electrophoresis for each of the tested patients with glomerulonephritis. 'Micro-sieving' can be used widely as a fundamental technique to analyze glomeruli in renal needle biopsy samples. Copyright (C) 2010 S. Karger AG, Basel

The seasonal variation in protein expression in the sporophyte of Saccharina japonica (Areschoug) Lane, Mays, Druehl and Saunders was investigated. High-quality proteins that are available for protein profiling were extracted by the ethanol/phenol extraction method, and 564 protein spots in total were detected. Proteins were identified through database search by combining Mascot and MS BLAST for 100 spots, and significant difference of expression level between the samples collected in winter and in summer was observed in the case of 95 spots. Within 67 spots upregulated in the samples collected in summer, vanadium-dependent bromoperoxidase (vBPO) were identified for 21spots. It is thought that the elevation of expression level of vBPO in summer depend on the activation of the functions: (1) elimination of active oxygen species and protection of the algal body from oxygen injury, (2) prevention of the growth inhibition due to the adherence of attached organisms, in the season.

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of alpha 1 and alpha 2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the alpha 1 chain, pepsin cleaved between Asp(1035) and Phe(1036), and actinidain between Gly(1032) and Gly(1033). Thus, the actinidain-hydrolyzed alpha 1 chain is shorter at the C terminus by three residues, Gly(1033), Phe(1034), and Asp(1035). In the alpha 2 chain, both proteases cleaved between Glu(1030) and Val(1031). We demonstrated that a synthetic nonapeptide mimicking the alpha 1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the alpha 1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.

Effective biomarkers for discrimination between ulcerative colitis (UC) and Crohn's disease (CD) have not been established yet. In this study, we analyzed protein profiles of peripheral blood mononuclear cells (PBMCs) of the patients to find such a biomarker. Peripheral blood mononuclear cell proteins from 17 UC patients, 13 CD patients, and 17 healthy controls were separated by two-dimensional gel electrophoresis. The intensities of individual protein spots were subjected to discriminant analysis of UC and CD using the SIMCA-P+program. We found that 547 protein spots were commonly detected among the UC, CD, and healthy groups. Orthogonal partial least squares-discriminant analysis using 276 protein spots clearly discriminated the UC patients from the CD patients (R (2) 0.994; Q (2) 0.462). A similar analysis using a further selected 58 protein spots showed higher performance for discrimination of the diseases (R (2) 0.948; Q (2) 0.566). Eleven out of the 58 protein spots were successfully identified; these were functionally related to inflammation, oxidation/reduction, the cytoskeleton, endocytotic trafficking, and transcription. In addition, the PBMC protein profiles were useful for the prediction of disease activity in the UC and the CD patients, and they were also useful for predicting disease severity and responses to treatments in the UC patients. PBMC protein profiles are useful for the discrimination of UC from CD. The profiles could be a potent biomarker for the differential diagnosis of these diseases. Further investigation of the proteins which contributed to the discrimination could promote elucidation of the pathophysiology of UC and CD.

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus. Lupus (2010) 19, 717-726.

We analyzed serum short peptides comprehensively to know whether they were useful to characterize IgA nephropathy (IgAN). Serum samples from 26 patients with untreated IgAN and 25 healthy donors were tested. Short peptides with molecular weights of similar to 7kDa, purified from the serum samples by magnetic-beads-based weak cation exchange, were detected by mass spectrometry. Then the peptide peaks detected were subjected to the multivariate data analysis by SIMCA-P+(R) containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA). A total of 92 peptide peaks were detected in the tested serum samples. The OPLS-DA analysis revealed that the profile of all the peptide peak intensities discriminated the IgAN group and the healthy group completely with a high R2 value (0.919) and a high Q2 value (0.861). Further, the profile of only five peptide peaks was found to discriminate the two groups. By tandem mass spectrometry and database searching, three of the five peptides which increased in the IgAN group were identified as fragments of fibrinogen alpha chain, and the two peptides which increased in the healthy group were identified as fragments of complement C3f and kininogen-1 light chain. Taken together, the profile of the serum short peptides would be useful to discriminate IgAN and healthy conditions. Further, the five peptides may be candidate serum markers for IgAN and may be related to pathogenesis of IgA. Copyright (c) 2009 John Wiley & Sons, Ltd.

Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function.

【目的】 現在、IgA腎症の確定診断は、腎炎症候に加え、腎生検でのメサンギウムにおけるIgA免疫複合体の沈着の確認である。しかし、腎生検は患者の身体的、精神的な苦痛を伴う。そのため、より簡便で侵襲性の低い診断方法の確立が求められている。今回、IgA腎症の診断マーカーとなりうる血中ペプチドの探索を目的として、未治療のIgA腎症患者の血清を用いた網羅的なペプチドミクス解析を行った。【方法】26名のIgA腎症患者および25名の健常者の血清から、弱陽イオン交換磁性ビーズ（MB-WCX, Bruker Daltnics社）を用いてペプチド（～7kDa）を回収し、MALDI-TOFによって検出した。次に、検出されたペプチドイオンのピーク面積データを用いて、Orthogonal-partial-least-squares判別分析(OPLS-DA)を実行し、IgA腎症患者群と健常人群を判別するモデルの作成を試みた。作成されたOPLD-DAモデルの判別予測力は、7分割の交差検定にもとづいたQ2値によって評価した。[結果と考察] MALDI-TOF解析によって、IgA腎症患者および健常人の血清から97本のペプチドが検出された。これらのペプチドの定量情報を用いてOPLS-DA解析を試みたところ、IgA腎症患者群と健常人群を完全に分離する予測力の高い判別モデルが作成された（R2 = 0.92, Q2 = 0.861）。97本のペプチドの内、IgA腎症患者群で有意に増加する5本 (m/z 2951.24, 5907.91, 5865.34, 3240.43, 2659.80, p < 0.01)と、有意に減少する1本（m/z 1778.80, p < 0.01）のペプチドが、両群の判別に特に有効であることが示され、この6本のペプチドの定量データのみを用いても、十分に予測力の高い判別モデルを作成することが出来た（sensitivity = 0.96, specificity = 0.96, R2 = 0.807, Q2 =0.790）。これら6本のペプチドはIgA腎症の診断マーカーとして有用であると考えられた。現在、これらマーカー候補ペプチドの同定および、IgA腎症の病態との関わりについての解析を進めている。

【背景と目的】全身性エリテマトーデス（SLE）は、多臓器を障害する自己免疫性疾患である。中でも、中枢神経に障害が生じる状態を中枢神経ループス（CNS-Lupus）とよび、診断が困難で非常に予後の悪い病態である。近年、このCNS-Lupus患者血清に神経細胞に対する自己抗体（抗神経細胞抗体）の存在することが報告された。しかし、抗神経細胞抗体の対応抗原はいまだ同定されていない。その為、本研究ではこれらを明らかにすることを目的とする。【方法と結果】培養したヒト神経芽細胞種株（SK-N-MC）より抽出したタンパク質をSDS-PAGE (1DE) にて泳動し、PVDF膜に転写した。この膜にCNS-Lupus患者30名及び中枢神経症状のないSLE患者30名の血清を各々反応させ、反応した抗原タンパク質を2群間で比較した。その結果、CNS-Lupus群で有意に反応が多いバンドが4つ認められた。次に、抗原タンパク質の同定を行うため、SK-N-MCの抽出タンパク質を2次元電気泳動（２DE）にて分離し、PVDF膜に転写した。CNS-Lupus患者群から目的抗原バンドに強く反応する患者血清5名分を混合してウェスタンブロットを行った。CNS-Lupus群で有意に反応が認められた１DEの4つのバンドの内、3つのバンドに相当するスポットを7つ検出した。それらのスポットのタンパク質をMALDI-TOF/MSにより同定した。その結果、 heat shock protein beta-1, peroxiredoxin-4, NADH dehydrogenase ubiquinone iron-sulfur protein 3, ubiquitin carboxyl-terminal hydrolase isoform L1, Histone H2A type 1, mitochondrial single-stranded DNA-binding protein の計6種のタンパク質を得た。【結語】今回我々は、CNS-Lupusにおける抗神経細胞抗体の対応抗原の候補タンパク質を6種同定した。現在、これらのタンパク質に対する抗体がCNS-Lupus患者血清で有意に高率に認められるか否かについての組換えタンパク質によるウェスタンブロットで検証を行っている。

〔背景と目的〕ヒトのめまいのモデル動物として片側内耳破壊（unilateral labyrinthectomy: UL）ラットが使用されている。ULにより、前庭神経核の活動性に左右差が生じ、その結果、前庭－動眼反射を介する眼振と、前庭－脊髄反射を介する平衡障害が出現する。しかし、時間の経過とともにこれらの症状は寛解し、この自然寛解を前庭代償と呼ぶ。これには、前庭神経核を中心とした中枢前庭系の情報伝達の再構築が関与しているが、その機序は未だ解明されていない。本実験は、前庭神経核に対し抑制性に働いて左右のアンバランスを是正していると考えられる小脳片葉の蛋白質の変化を網羅的に解析することにより、めまいの治療標的となりうる蛋白質を探索することを目的としている。〔方法と結果〕 Sprague Dawley ラットにULを施行し、前庭代償モデルを作成した。前庭代償急性期（48時間）と慢性期（1週間）に小脳片葉（破壊側、対側）を摘出し、蛋白質を抽出した。これを2次元ディファレンシャルゲル電気泳動で分離して解析した。48時間後および1週間後のUL群（破壊側、対側）をsham群（手術側、対側）とそれぞれ比較したところ、のべ120個のスポットが、UL群で有意に増加（1.3倍以上）もしくは減少（1/1.3倍以下）していた。その内21個のスポットが手術側、対側、48時間後、1週間後のいずれかで重複しており、結果、のべ99個の蛋白質スポットが変化していた。この99個中、48時間後において、両側小脳片葉で有意に変化したのが4個、片側のみで有意に変化したのが45個、さらに48時間後に変化せず1週間後にのみ変化するスポットが50個（破壊側のみで変化21個、対側のみで変化25個）であった。片側のみ変化の45個のうち30個は内耳破壊対側で変化しており、さらにそのうち19個が1週間後に両側で変化なしとなった。すなわち、48時間後対側でのみ変化する19個のスポットは急性期の代償に、1週間後に変化する50個のスポットは慢性期の代償に深く関与していると考えられる。次にこれらのスポットについてMALDI-TOF MSを用いて同定を試みた。その結果、13スポットから11種類の蛋白質、vesicle-fusing ATPase, mitochondrial aconitate hydratase, brain glycogen phosphorylase, heat shock congnate 71 kDa protein, lamin-B1, alpha-internexin, heterogeneous nuclear ribonucleoprotein K, protein disulfideisomerase A3, mitochondrial ATP synthase subunit beta, mitochondrial ATP synthase subunit b を同定できた。〔結語〕 前庭代償急性期には、対側の小脳片葉が、慢性期には両側の小脳片葉が関与している可能性が示唆された。今後、前庭代償に関与するスポットを同定し、同定した蛋白質については機能解析を行い、めまいの治療標的分子を解明していく予定である。

【背景と目的】
関節リウマチ（RA）は、病因解明と根治療法が求められている難治性疾患である。RAでは、最近シトルリン化タンパク質に対する自己抗体が疾患マーカーとなることが判明し、タンパク質のシトルリン化がRA病態に関与する可能性が示唆されている。しかし、シトルリン化以外の翻訳後修飾（PTM）がRAに関与しているか否かについてはほとんど報告がなく、調べられていないのが現状である。そこで本研究では、PTMの1つである「アセチル化」に着目し、健常者に較べRA患者で有意に強くアセチル化されているタンパク質を網羅的に検出・同定することで、RAに関連するアセチル化タンパク質が存在するか否かを検討した。なお、PTMの網羅的探索は、リン酸化や糖鎖を除いては一般に進んでおらず、アセチル化タンパク質の網羅的解析法の確立はその一助となる。
【方法】
RA患者および健常者より、末梢血リンパ球由来タンパク質を調製し、二次元電気泳動により展開した。ゲル内タンパク質をSypro Rubyにて染色後、PVDF膜へ転写した。Sypro Rubyによりタンパク質スポットのゲル画像を取得後、さらに抗アセチル化リジン抗体を用いてウェスタンブロッティングを行い、アセチル化タンパク質スポットを検出した。RA患者と健常者間で、アセチル化量に差異のあったタンパク質スポットについて、アセチル化タンパク質スポット画像とSypro Rubyでのタンパク質スポット画像を重ねることで、その位置を確定した。そのタンパク質をトリプシンでゲル内消化し、回収後MALDI-TOF/TOF型質量分析により同定した。
【結果】
健常者に較べ、調べたRA患者に共通してアセチル化が顕著に亢進しているタンパク質スポットが複数個見出された。それらタンパク質スポットのうち2つはMALDI-TOF/TOF型質量分析により、&alpha-エノラーゼ（ENO1）とイソクエン酸脱水素酵素１（ICDH1）であると同定された。これらのアセチル化が、どのようにRAに関与しているのか明らかにするために、現在、同タンパク質の酵素活性とアセチル化の関係をはじめとした解析を行なっている。
【結語】
RAにおいて、ENO1とICDH1のアセチル化が顕著に亢進していることから、それらのアセチル化が、RAの疾患マーカーとして機能する可能性、あるいは、それらがアセチル化を受けることによる構造もしくは機能変換が、病態プロセスに関与する可能性が示された。

Unilateral labyrinthectomy (UL) in rats is used as a human vertigo model. In this model, spontaneous nystagmus and dysequilibrium caused by UL are ameliorated within 48-72 hours. The amelioration, termed vestibular compensation (VC), is long lasting. Although cerebellar flocculi have been reported to be involved in VC, the molecular mechanisms behind VC are unknown. In this study, we used 2D-DIGE to detect protein changes in flocculi during acute (48 hours) and chronic (1 week) stages of VC. We found 99 out of 967 protein spots that showed significant changes in their intensities. Of the 99 spots, 45 spots (ipsilateral side, 15; contralateral side, 30) changed unilaterally during the acute stage, whereas 46 spots (ipsilateral side, 21; contralateral side, 25) changed unilaterally during the chronic stage. Thus, the acute compensation mechanism is more complicated in the contralateral flocculus than in the ipsilateral flocculus. Using MALDI-TOF MS, we identified 10 proteins out of the 12 protein spots. Of these, 3 proteins involved in synaptic transmission, neuronal filament formation and vesicular transport, respectively, demonstrated VC generation.

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.

A novel 19 kDa protein, which was named N19, was isolated from the nacreous layer of the pearl oyster Pinetada fucata. N19 is one of predominant proteins found in the water-insoluble fraction of the nacreous layer. MALDI-TOF/TOF analysis indicated that the three trypsin-digested peptides (791.45, 824.42, and 1118.65 m/z) corresponded to the amino acid sequences predicted from a cDNA isolated from a mantle cDNA library of P. fucata. Northern blot analysis revealed that the N19 mRNA was a little more abundant in the pallial region than the edge region, in the mantle. In CaCO3 precipitation assay, the recombinant N19 protein inhibited the crystallization of CaCO3. These results indicate that N19 is localized in the nacre and plays a negative regulatory role in calcification in the pearl oyster. (C) 2007 Elsevier Inc. All rights reserved.

In molluscan shellfish, pigmentation is frequently observed in the calcified shell, but the molecular basis of this process is not understood. Here, we report two tyrosinase proteins (Pfty1 and Pfty2) found in the prismatic shell layer of the pearl oyster Pinctada fucata; this layer is recognized as the pigmented region in P. fucata. The protein sequences were deduced from the corresponding cDNAs and confirmed by MALDI-TOF/TOF analysis. The sequences suggest that both tyrosinases have two copper-binding sites in similar N-terminal domains that are homologous to tyrosinases of cephalopods and hemocyanins of gastropods. In turn, this suggests that bivalve tyrosinases are evolved from a common ancestral copper-binding protein in the mollusc. Pfty1 and Pfty2 were specifically expressed in the mantle, and their expression in the mantle is different from each other, suggesting that these tyrosinases have distinctive roles in melanogenesis in shells. (c) 2006 Elsevier Inc. All rights reserved.

Random sequencing of molecules from a cDNA library constructed from mantle mRNA of the pearl oyster Pinctada fucata was used to obtain information on organic matrix proteins in the shell. In the determined sequences, we identified 7 distinct cDNAs encoding similar glycine-rich domains. Complete sequence analysis of these cDNAs showed that the predicted sequences of the proteins, which we named shematrins, possessed similar domains comprising repeat sequences of two or more glycines, followed by a hydrophobic amino acid. In addition, in shematrin-1, -2 and -3, a repeat domain designated as XG(n)X (where X is a hydrophobic amino acid) was conserved. It is of further note that all the shematrin proteins have RKKKY, RRKKY or RRRKY as their C-terminal sequence. According to northern blot analysis, all shematrins are exclusively expressed in the mantle, and particularly in the edge region of the mantle-, furthermore, peptide fragments similar to shematrin-1 and -2 were detected in the prismatic layer of shells by MALDI-TOF/TOF MS analysis. These findings suggest that many of shematrins are synthesized in the mantle edge and secreted into the prismatic layer of the shell, where the protein family is thought to provide a framework for calcification. (c) 2006 Elsevier Inc. All rights reserved.

The reaction mechanism of the coagulation of soy protein isolates (SPIs) induced by subtilisin Carlsberg was investigated. Formation of the coagula was monitored by measuring the turbidity (OD660) of the SPI solution, which decreased at the initial stage (phase 1 or digestion phase) of the reaction, and then increased (phase 2 or coagulation phase) and finally reached the plateau level. The velocity of the coagulation increased with increasing enzyme concentration. The coagulation was inhibited dramatically by adding a serine protease inhibitor (phenylmethanesulfonyl fluoride, PMSF) when the turbidity reached the minimum value. This indicates that the SPI digests participating in the coagulation are produced mainly in phase 2; in other words, production of the coagulating fragments and their coagulation occur simultaneously in phase 2. Structural changes of SPI during proteolysis were measured by observing fluorescence changes of aromatic amino acids of SPI and an externally added hydrophobic probe. It was suggested that the hydrophilic surface areas of SPIs might be cleaved preferentially in phase 1, and that the hydrophobic inner areas might be cleaved in phase 2 with extensive decomposition of the 3-D structure of SPI proteins. The fragments formed in phase 2 are considered to coagulate through hydrophobic interactions.

The coagulation of soy protein isolates (SPI) induced by subtilisin Carlsberg was studied. The proteins were digested to fragments of 16 kDa or less in the early stage of the reaction, followed by coagulation. The time-course of the coagulation measured by turbidity was separated into three phases. The turbidity decreased from the initial level observed at time zero to the minimum level (OD1) at time T1 (15-20 min) in the first phase. Then, it increased drastically to reach the maximum (OD2) at time T2 (60-70 min) in the second phase, which was followed by a slight decrease in the third phase. The coagulation was terminated at T2, where 30-35% of the weight of the SPI proteins was in coagula. Proteins in the coagula were degraded slowly in the prolonged incubation, and the protein content in the coagula was finally 15-20% of the weight. The time-course of the turbidity agreed well with that of the weight of the precipitates formed, indicating that the turbidity reflects the progress of the coagulation. The turbidity change (OD1 to OD2) from the start to the end of the coagulation increased proportionally to the SPI concentration (4.9-11 mg/mL), although the time (T1 to T2) needed for the coagulation was independent of the concentration. The growth of the coagula is promoted by increasing the SPI concentration and is rate-limiting in the coagulation.

Myeloperoxidase (MPO) is one of the major autoantigens of ANCA-associated vasculitis. We have shown that immunization of mice with oxidized murine MPO in vitro results in the production of autoantibodies against normal MPO. In this presentation, we show that immunization of mice with mouse serum albumin (MSA) with oxidative modifications produces autoantibodies in a similar manner, indicating that the phenomenon that immunization with an oxidized abnormal protein promotes the production of autoantibodies may be a universal phenomenon.

To elucidate the molecular mechanism of the anti-inflammatory effect of mango leaf extract (MLE), LPS-activated Raw264.7 cells were treated with MLE and subjected to quantitative proteomic analysis and Western blot analysis of p62, an autophagy marker. The results showed that MLE promoted early activation of autophagy.

Type 1 collagen from pig tendon or dermis was analyzed by LC-MS/MS for comprehensive quantitative analysis of post-translational modifications. 88 post-translational modifications were identified. Of these, 49 were classified as enzymatic modifications, many of which have not been previously reported. Of these, G-HyP-HyP, hydroxylysine (HyK), phosphorylation, and glycosylation, which have been reported to be involved in collagen fiber properties, were identified as sites of modification by verifying MS/MS spectra. sites of G-HyP-HyP and phosphorylation were more common in tendon for both alpha1 and alpha2 chains The sites of modification were more common in tendon. Although the number of HyK and glycosylation sites were similar between the tissues, many sites, including K354, K450, K507, K783 in α1 chains and K177 in α2 chains, were detected in tendon. K354, K450, K507, K783, and K177 of the α2 chain. In addition, quantitative comparison of glycans using XIC clearly showed that glycosylation to 87K of α1 chain and 174K or 182N of α2 chain was more frequent in dermis than in tendon, and conversely, glycosylation to 408K of α1 chain was more frequent in tendon. These differences in PTMs may give rise to the characteristics of collagen fibers in tendon from tissue to tissue.

Recently, we have shown that an ethanol extract of mango leaves (MLE) has strong anti-inflammatory effects in a system using macrophage-like Raw264 cells activated with lipopolysaccharide (LPS) from Escherichia coli. In this study, we attempted to elucidate the mechanism of action of MLE by measuring changes over time in the protein profile of LPS-activated Raw264.7 cells treated with MLE using the SWATH method, and comparing LPS- and LPS+ groups after 24 hours of treatment, we found a decrease in proinflammatory factors such as ERK and p-38 MAPK In the LPS- and LPS+ groups, there was a decrease in proinflammatory factors such as ERK and p-38 MAPK and an increase in proinflammatory factors such as SQSTM1. Principal component analysis of quantitative data at 6 hours of LPS treatment showed that enzymes related to glycolysis/anaerobic respiration decreased and proteins related to mitochondrial function increased in the MLE group. Since it is known that decreased mitochondrial function exacerbates inflammation through activation of the NLRP3 inflammasome, the results indicate that MLE may exert its anti-inflammatory effects by working toward maintaining mitochondrial function homeostasis.

In this study, type I collagen extracted from porcine dermis and tendon was analyzed by nanoLC-MS/MS, and quantitative inter-tissue comparison of 3HyP was attempted by comprehensive post-translational modification analysis and semi-quantitative analysis based on PTM detection frequency. Collagen extracted from pig dermis and tendon was trypsin digested in solution, and the resulting peptide fragments were analyzed by nanoLC-MS/MS (TripleTOF5600+, SCIEX). Exhaustive post-translational modification searches revealed more HyP sites than previously reported: 174 in dermis and 121 in tendon for the α1 chain, and 144 in dermis and 155 in tendon for the α2 chain. Furthermore, by examining the MS/MS spectra, we identified 26 G-HyP-HyP sites in the α1 chain and 14 G-HyP-HyP sites in the α2 chain. Of these, 39 were novel sites not previously reported. Comparing dermis and tendon, the number of G-HyP-HyP sites in the α1 chain was 13 in dermis versus 23 in tendon, and in the α2 chain, 6 in dermis versus 11 in tendon. In particular, the frequency of detection of 3HyP in α1 chain Pro14 was clearly higher in tendon (14% vs. 0.89% in dermis), and similarly, the frequency of detection of 3HyP in α2 chain Pro707 was also clearly higher in tendon (41% vs. 13% in dermis). These tendon-specific 3HyPs may give rise to the collagen fiber characteristics of tendon.

以下の2つの実験を実施した。 実験1 SD系雄ラットに①Glu＋大豆油、②Fru＋大豆油、③Glu＋魚油、④Fru＋魚油を含む飼料を4週間給餌し、各種パラメーターを解析した。肝臓重量はFru群で有意に高く、副睾丸脂肪および腎周囲脂肪重量は魚油群で有意に低かった。血漿トリグリセリド濃度は交互作用がある傾向が見られ、他の3群と比較してFru＋大豆油群で有意に高くなった。肝臓ではFasn、Acacaの発現がFru＋大豆油群で有意に高く、Acox1の発現は魚油群で有意に高かった。腸間膜脂肪ではAcox1の発現は魚油群で有意に低かった。また、Lpl発現はFru＋魚油群が他の3群と比較して有意に低かった。以上の結果より、魚油の作用としてよく知られている脂質代謝改善作用は高Fru食給餌下でも観察されるが、腸間膜脂肪では脂肪酸酸化はむしろ低下していることが分かった。 実験2 SD系雄ラットに①Glu＋ラード、②Fru＋ラード、③Glu＋中鎖脂肪、④Fru＋中鎖脂肪を含む飼料を4週間給餌し、各種パラメーターを解析した。最終体重は、ラード群に高い傾向が見られた。血漿中Glu濃度はMCT群、Fru群で有意に高く、血漿中トリグリセリド(TG)濃度はラード群、Fru群で有意に高かった。肝臓重量はFru群が有意に高く、Acaca，Fasn等の脂肪酸合成系関連遺伝子発現はMCT群で有意に高かった。腸間膜脂肪重量に差は見られなかったが、Fasn, Scd1, Acaca, Me1, Chrebp等の脂肪酸合成系関連遺伝子発現がMCT群、Glu群で有意に高かった。これらの結果から、腸間膜脂肪では、Fruによる脂肪合成促進作用、MCTによる脂肪酸酸化作用は見られず、むしろGluまたはMCTで脂肪酸合成関連遺伝子発現が高まり、一方で腸間膜脂肪重量には影響しないことが分かった。

令和2年度は脂肪と糖質の割合のみが異なる普通食（ND）と高脂肪食（HFD）をC57BL/6Jマウスに5週間摂取させて初期のメタボリックシンドロームの状態に誘導し、16時間の絶食の後に採血と肝臓、脂肪組織、小腸組織の採取を行った。このうち、肝臓の定量プロテオミクス解析を実施し、絶食時には、脂肪組織から流入した脂肪の分解、アセチルCoAからのケトン体の合成、TCA回路の抑制と解糖系の更新などが起こることが知られているが、HFD群ではND群に比べて、①脂肪酸のβ酸化、②ケトン体やコレステロールの合成に関わる酵素の量が増加した。ここから、極初期のメタボリックシンドロームでは糖新生の進行が抑制されており、脂肪分解で生じたグリセロールからピルビン酸に至る経路上の代謝物が蓄積している可能性が考えられた。 令和3年度は、NDとHFDをC57BL/6Jマウスに同じく5週間摂取させた後、最終日に絶食を行わずに、両群共にND食を摂取させることで条件をそろえ、昨年度と同様に肝臓の定量プロテオミクス解析を行った。その結果、本条件では、ND群とHFD群間では肝臓プロテオームプロファイルに大きな違いは存在せず、脂肪酸分解や合成反応に関する酵素など脂肪の蓄積量を反映した変化しか見いだせなかった。ここから、メタボリックシンドロームにおける代謝異常は空腹時における異常が引き金となっている可能性が示唆された。 また、令和3年度は、他に高脂肪食の摂取期間を10週に伸ばして同様の実験を行い、肝臓プロテオームの測定を実施しており、現在解析を進めている。他に令和2年度3年度に採取した脂肪組織や小腸組織のプロテオーム測定も進めデータの蓄積を行っている。

In this study, we analyzed post-translational modifications (PTMs) of autoantigen Myeloperoxinase (MPO) in peripheral blood neutrophils from patients with ANCA-related vasculitis. Neutrophil MPO was purified from peripheral blood of 8 patients with ANCA-related vasculitis and 8 healthy controls, digested with trypsin, and analyzed by high-performance LC-MS. Database search against human proteome database identified 52 PTMs on the MPO molecules. Among them, multiple post-translational modifications such as Carbamylation were significantly increased in the patient group. Furthermore, the immunization of mice with a post-translational modification-introduced mouse protein causes a temporary increase in the antibody titer against the self-protein. These results indicated the relationship between abnormal post-translational modification and autoantibody production.

Serum peptides which are components of novel biomarker candidates for dementia with Lewy bodies(DLB) were identified and quantified, and their effects on neural cells were examined. One of the peptides, peak of which was at 4090 m/z, was identified as a fragment of a coagulation factor. We prepared internal control peptides labeled with stable isotopes and quantified the peptides, peaks of which were at 1737 m/z, 2898 m/z, 4052 m/z, and 4090 m/z, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). High linearity was obtained in all the peptide quantification in the range of less than 20 pmol/μL (R2>0.96). Thus, it was suggested that the peptides are accurately quantified by MALDI-TOF/MS using internal standard peptides. Furthermore, those peptides affected cytokine profiles of human hippocampal cells, which indicates that the peptides may be associated with the pathophysiology of DLB and would be a therapeutic target for the disease.

In this study, relations between carcass traits and exosomal miRNA expression profiles in serum were analyzed to identify circulating exosomal miRNA biomarkers for predicting carcass traits and meat quality characteristics of Japanese Black cattle by bioinformatics analysis. Eleven secreted microRNAs were listed as biomarker candidates as a result of microRNA microarray analysis using serum of fattening cattle. Subsequently, reproducibility was confirmed for the two biomarker candidate microRNAs by the association analysis between the amount of blood microRNA quantified by real-time PCR analysis and the carcass traits. Those selected microRNAs were inferred to affect the expression of genes related to differentiation and development of cells constituting muscle tissue by computational searches of existing public databases.

In this study, to identify circulating exosomal miRNA biomarkers for predicting carcass traits and meat quality characteristics of Japanese Black cattle by bioinformatics analysis, we first compared exosomal miRNA expression profiles of serum between low and high merit steers in main carcass traits. We then inferred the involvement of the lower- or higher-expressed exosomal miRNAs in metabolic pathways by computational searches of existing public databases (miRDB, and PANTHER). As a result, our study suggests the possibility of at least eight identified exosomal miRNAs as biomarker candidates for predicting carcass traits and meat quality characteristics during fattening of Japanese Black cattle.

It was studied whether‘measurement method of splicing activity of peripheral blood mononuclear cells (PBMCs)’is useful as a single diagnosis of systemic lupus erythematosus (SLE). Firstly, the measurement method of splicing activity of PBMCs was developed and optimized. Next, the splicing activity of PBMCs from the patients with SLE and healthy donors was measeured by this methods. As a result of that, there was no difference on the splicing activity between groups of the patients with SLE and healthy donors. For a while, it was found that the measurement of splicing activity of only T cells may be more accurate than that of PBMCs, so I will investigate splicing activity of T cells from the patients with SLE and healthy donors.

In this study, we tried to establish an useful method for detecting a de-phosphorilated form of U1-68k autoimmunogen, which was found to be increased in peripheral blood mononuclear cells of patients of systemic lupus erythematosus or Mixed connective tissue diseases. As a substitution of a conventional 2D-western blot method, we examined three methods; (1) an immunological method, (2) a phos-tag electrophorsis, and (3)an isoelectric focusing electrophoresis-western blot. As a result, we found that the isoelectric focusing electrophoresis-western blot method is most suitable for the detection of the de-phosphorilated form of U1-68k. Moreover, we found that whole cell extract prepared by cell lysis buffer containing SDS can be used for detecting U1-68k. In conclusion, combination of isoelectric focusing electrophoresis-western blot and whole cell extract by SDS-containing cell lysis buffer can shorten analsys time from 5 days to 2.5 days.

Post-translational modifications (PTMs) are often critical for diagnosis of diseases. Thereby, we here tried to elucidate alteration of PTMs in RA, focusing on acetylation in this study. We applied acetyl-proteomics to peripheral blood mononuclear cells (PBMCs) to elucidate PTM difference between patients with RA and healthy. Multiple proteins in PBMCs were highly acetylated in the RA groups. One of the proteins predominantly acetylated in the RA group was identified to be ENO1. Next, we tried to identified acetylated lysine residues, but we could not detect RA-specific acetylated peptides of ENO1. This would be technical limitation of mass spectrometric analysis at present. In the future, it would be needed to establish more effective methods to identify acetylated lysine residues. Next, we examined whether acetylation affect the antigenecity of ENO1 in patients with RA. As a result, the reactivity was not different between the acetylated ENO1 and the non-acetylated ENO1.