Kouhei Nagai

A novel nacre protein N19 in the pearl oyster Pinctada fucata

A novel 19kDa protein, which was named N19, was isolated from the nacreous layer of the pearl oyster Pinctada 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 1118.65m/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 CaCO(3) precipitation assay, the recombinant N19 protein inhibited the crystallization of CaCO(3). These results indicate that N19 is localized in the nacre and plays a negative regulatory role in calcification in the pearl oyster.

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

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.

Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): Recalcitrant tissue containing high levels of viscous polysaccharides

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.

Seasonal changes in proteomic profiles of Japanese kelp: Saccharina japonica (Laminariales, Phaeophyceae)

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.

Comprehensive analysis of short peptides in sera from patients with IgA nephropathy

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 approximately 7 kDa, 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+ 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.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement

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.

Temperature stress-induced changes in the proteomic profiles of Ecklonia cava (Laminariales, Phaeophyceae)

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 15°C (control) to 10°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 20°C. With the cultivation at 30°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 30°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.

Arthritogenicity of annexin VII revealed by phosphoproteomics of rheumatoid synoviocytes

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 TNFα 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.

Roles of serum fibrinogen α chain‐derived peptides in Alzheimer's disease

To find a blood biomarker and disease‐related peptides in Alzheimers disease (AD), we comprehensively detected serum peptides.

Proteomic analysis of the rat cerebellar flocculus during vestibular compensation

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 altered expression only in the acute stage. Our results enhance the understanding of the role of the cerebellar flocculi in VC generation.