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1.  A novel indole compound MA-35 attenuates renal fibrosis by inhibiting both TNF-α and TGF-β1 pathways 
Scientific Reports  2017;7:1884.
Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) play key roles in progression of renal fibrosis, dual blockade of TGF-β1 and TNF-α is desired as its therapeutic approach. Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5-(3, 5-dimethoxybenzyloxy)-3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-β1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-β1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-β1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis.
PMCID: PMC5432497  PMID: 28507324
2.  Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases 
EBioMedicine  2017;20:27-38.
Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model “Mitomouse” (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.
Graphical Abstract
Image 1
•MA-5 rescues patient's cell survival independent of genetic mutation backgrounds.•MA-5 promotes ATP synthase dimer formation followed by the precipitation of supercomplex.•The cell protective effect of MA-5 will be predicted by GDF-15 in vitro and in vivo.
Mitochondrial diseases are life-threatening and progressive, yet largely untreatable because of the lack of adequate drug. We found a mitochondria-homing drug Mitochonic acid-5 (MA-5) that facilitates ATP production and restoring mitochondrial dynamics, rescuing a wide variety of human mitochondrial diseases cell survival. MA-5 binds to mitochondrial protein mitofilin/Mic60 and facilitating ATP synthase oligomerization and supercomplex formation that increase local ATP production, even under the genetically proton-limited condition in mitochondrial diseases.
Therefore, MA-5 will be a candidate chemical for modulating mitochondrial function and remedy for not only mitochondrial diseases but also mitochondrial-related diseases (diabetes, diabetic nephropathy, cardiomyopathy, longevity etc.).
PMCID: PMC5478234  PMID: 28579242
MA-5, 4-(2,4-difluorophenyl)-2-(1H-indole-3-yl)-4-oxobutanoic acid; MELAS, myopathy encephalopathy lactic acidosis and stroke-like episodes; KSS, Kearns-Sayre syndrome; LHON, Leber hereditary optic neuropathy; ETC, electron transfer complex; BSO, l-buthionine-(S,R)-sulfoximine; CPEO, chronic progressive external ophthalmoplegia; OCR, oxygen consumption rate; ECAR, extra-cellular acidification rate; OXPHOS, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; MINOS, mitofilin/mitochondrial inner membrane organizing system; MA-5; Mitochondrial disease; ATPase dimer formation; Supercomplex; GDF-15
3.  Chronic Encapsulated Intracerebral Hematoma: Endoscopic Removal as Minimally Invasive Surgery for a Patient with Alcoholic Cirrhosis 
NMC case report journal  2017;4(2):51-53.
Chronic encapsulated intracerebral hematoma (CEIH) is a rare cerebrovascular disease. Here, we describe a case of a patient with alcoholic cirrhosis and an abnormal coagulation state that was treated with minimally invasive endoscopic hematoma removal. A 54-year-old man presented with a 2-week history of incoherent speech, incontinence, and repeated stumbling. Laboratory analysis showed thrombocytopenia and the prolongation of prothrombin time due to alcoholic cirrhosis. Computed tomography and magnetic resonance imaging showed a large mass in the left parieto-occipital lobe suggesting a hematoma. Minimally invasive endoscopic hematoma removal was then performed. CEIH was diagnosed from the intraoperative findings of serous hematoma, a thin yellowish capsule, and old clots. The postoperative course was uneventful and there was no hematoma recurrence. In conclusion, endoscopic hematoma removal may be one of the options for the treatment of CEIH in patients with cirrhosis and an abnormal coagulation state.
PMCID: PMC5453300
chronic encapsulated intracerebral hematoma; endoscopy; minimally invasive surgery
4.  Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator 
The Journal of Experimental Medicine  2016;213(8):1441-1458.
mTEC progenitors differentiate to mature Aire+ mTECs through a pathway that initiates with RANK and LtβR signaling via the nonclassical NF-κB, followed by TRAF-6–driven maturation.
Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs.
PMCID: PMC4986530  PMID: 27401343
5.  Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x)) 
Breeding Science  2016;66(2):234-243.
Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes.
PMCID: PMC4785001  PMID: 27162495
Festuca arundinacea; festulolium; flow cytometry (FCM); fluorescence in situ hybridization (FISH); genomic in situ hybridization (GISH); Lolium multiflorum; DNA ploidy
6.  Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies 
Experimental Animals  2016;65(2):175-187.
The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module (“Kibo”) on the International Space Station. The CBEF provides “space-based controls” by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.
PMCID: PMC4873486  PMID: 26822934
habitat cage unit; microgravity; mouse; spaceflight
7.  Novel PLA2G6 mutations associated with an exonic deletion due to non-allelic homologous recombination in a patient with infantile neuroaxonal dystrophy 
Human Genome Variation  2015;2:15048-.
Novel PLA2G6 mutations associated with p.Asp283Asn and a unique intragenic deletion of exons 4 and 5 due to non-allelic homologous recombination were identified in a Japanese female patient with typical infantile neuroaxonal dystrophy. The patient showed progressive tetraplegia beginning at 9 months. An electroencephalogram showed a diffuse increase in fast waves, and brain magnetic resonance imaging showed progressive brain atrophy and T2 hypointensity in the globus pallidus.
PMCID: PMC4785535  PMID: 27081553
8.  Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice 
PLoS ONE  2015;10(10):e0141650.
Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear’s vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.
PMCID: PMC4626100  PMID: 26513242
9.  Roles of the membrane-reentrant β-hairpin-like loop of RseP protease in selective substrate cleavage 
eLife  null;4:e08928.
Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a β-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible β-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination.
eLife digest
Cells have communication systems that enable them to respond to potentially dangerous changes in their external environment. For example, bacteria have an enzyme called RseP that helps to activate responses to external stresses. This enzyme sits in the membrane that surrounds the cell and cuts a protein called RseA to release a signal molecule into the cell interior. This signal molecule then promotes the expression of particular genes to protect the cell from harm.
Previous studies have identified the ‘active site’ of RseP, which is the region of the enzyme that actually cuts the target protein. However, it is not clear how the enzyme is able to identify and cleave RseA and its other ‘substrate’ proteins. The enzyme also contains a structure called a β-hairpin-like loop that is close to the active site, which is not commonly found in membrane proteins. Here, Akiyama, Mizuno et al. used genetic and biochemical techniques to study the role of this loop structure in the RseP enzyme of the E. coli bacterium.
The experiments show that the loop specifically binds to a section of substrate proteins—called the transmembrane segment—that spans the cell membrane. Several genetic mutations that affected the loop altered the ability of RseP to bind to and cleave substrates. The effect of these mutations in RseP could be suppressed by introducing genetic mutations in substrates that altered the transmembrane segments. Akiyama, Mizuno et al. propose that the β-hairpin-like loop of the RseP enzyme binds the transmembrane segment of a substrate and presents it to the active site.
A previous study showed that another region of RseP called the periplasmic PDZ domains can act as a filter to stop RseP cutting other membrane proteins in error. Akiyama, Mizuno et al.'s findings suggest that the β-hairpin-like loop serves as an additional checkpoint to identify RseA and other proteins that RseP targets. The next step is to carry out further experiments to test this model.
PMCID: PMC4597795  PMID: 26447507
extracytoplasmic stress response; helix-destabilizing residue; small membrane protein; regulated intramembrane proteolysis; RpoE; E. coli
10.  Catalytic subunits of the phosphatase calcineurin interact with NF-κB-inducing kinase (NIK) and attenuate NIK-dependent gene expression 
Scientific Reports  2015;5:10758.
Nuclear factor (NF)-κB-inducing kinase (NIK) is a serine/threonine kinase that activates NF-κB pathways, thereby regulating a wide variety of immune systems. Aberrant NIK activation causes tumor malignancy, suggesting a requirement for precise regulation of NIK activity. To explore novel interacting proteins of NIK, we performed in vitro virus screening and identified the catalytic subunit Aα isoform of serine/threonine phosphatase calcineurin (CnAα) as a novel NIK-interacting protein. The interaction of NIK with CnAα in living cells was confirmed by co-immunoprecipitation. Calcineurin catalytic subunit Aβ isoform (CnAβ) also bound to NIK. Experiments using domain deletion mutants suggested that CnAα and CnAβ interact with both the kinase domain and C-terminal region of NIK. Moreover, the phosphatase domain of CnAα is responsible for the interaction with NIK. Intriguingly, we found that TRAF3, a critical regulator of NIK activity, also binds to CnAα and CnAβ. Depletion of CnAα and CnAβ significantly enhanced lymphotoxin-β receptor (LtβR)-mediated expression of the NIK-dependent gene Spi-B and activation of RelA and RelB, suggesting that CnAα and CnAβ attenuate NF-κB activation mediated by LtβR-NIK signaling. Overall, these findings suggest a possible role of CnAα and CnAβ in modifying NIK functions.
PMCID: PMC5377069  PMID: 26029823
11.  Porphyromonas gingivalis-derived Lysine Gingipain Enhances Osteoclast Differentiation Induced by Tumor Necrosis Factor-α and Interleukin-1β but Suppresses That by Interleukin-17A 
The Journal of Biological Chemistry  2014;289(22):15621-15630.
Background: We previously reported that Kgp, a lysine gingipain, degraded osteoprotegerin, an osteoclastogenesis inhibitory factor, to enhance lipopolysaccharide-induced osteoclastogenesis.
Results: Kgp enhanced tumor necrosis factor-α- and interleukin-1β-induced osteoclastogenesis.
Conclusion: Kgp degraded osteoprotegerin more efficiently than other cytokines, which might be related to enhancement of osteoclastogenesis by Kgp.
Significance: Degradation of osteoprotegerin may be a crucial event in periodontal osteolysis.
Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159–166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.
PMCID: PMC4140917  PMID: 24755218
Cell Differentiation; Cytokine; Inflammation; Osteoclast; Proteolytic Enzyme; Gingipain; Osteoprotegerin
12.  Limitation of immune tolerance–inducing thymic epithelial cell development by Spi-B–mediated negative feedback regulation 
The Journal of Experimental Medicine  2014;211(12):2425-2438.
Akiyama et al. show that transcription factor Spi-B is up-regulated by RANKL to trigger mTEC differentiation. Osteoprotegerin is also induced by this signaling pathway and acts as a negative feedback loop to attenuate mTEC development and thymic T reg cells.
Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B–mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage–specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL–Spi-B–OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.
PMCID: PMC4235644  PMID: 25385757
13.  Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells 
Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell–cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell–cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.
PMCID: PMC4568481  PMID: 26441966
medullary thymic epithelial cells; autoimmune disease; negative feedback; mathematical modeling; T cells; thymus
14.  Prevalence of anti-phospholipase A2 receptor antibodies in Japanese patients with membranous nephropathy 
Membranous nephropathy (MN) is the leading cause of nephrotic syndrome in adults. Anti-M-type phospholipase A2 receptor (anti-PLA2R) antibodies are found in most patients with idiopathic MN (iMN) worldwide, but the prevalence of anti-PLA2R antibodies among Japanese patients with MN is unknown. In this study, we determined the prevalence of anti-PLA2R antibodies in Japanese patients with MN.
The study population of our retrospective cross-sectional consisted of 131 patients with biopsy-proven MN who had not received any immunosuppressive treatments at time of both renal biopsy and serum sample collection. Of these, 100 had iMN and 31 had secondary MN (sMN). The circulating anti-PLA2R antibodies were analyzed using a highly sensitive Western blot analysis. Analysis was performed under non-reducing conditions with a human glomerular extract at serum dilutions of 1:25, 1:10, and 1 as the primary antibody.
Anti-PLA2R antibodies were detected in 53 (53 %) of 100 patients with iMN and 0 (0 %) of 31 patients with sMN. The prevalence of anti-PLA2R antibodies was higher in patients with nephrotic syndrome (61 %) than in patients without nephrotic syndrome (43 %). The number of patients with serum albumin ≤3.0 g/dL was significantly higher in those with anti-PLA2R antibodies (92 %) than that in those without them (68 %).
Anti-PLA2R antibodies were found in Japanese patients with iMN; however, the prevalence was lower than that of any other Asian country. This may indicate that the presence of other pathogenic antigens plays a significant role in Japanese patients with iMN.
PMCID: PMC4543411  PMID: 25412738
Phospholipase A2 receptor; Antibody; Membranous nephropathy; Prevalence; Japan; Western blot
15.  Classification of microbial α-amylases for food manufacturing using proteinase digestion 
Food Science & Nutrition  2014;2(5):571-577.
Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion–HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups.
PMCID: PMC4237487  PMID: 25473515
Endoproteinase Lys-C; HPLC; peptide; trypsin; α-amylase
16.  Mitochondria–Nucleus Shuttling FK506-Binding Protein 51 Interacts with TRAF Proteins and Facilitates the RIG-I-Like Receptor-Mediated Expression of Type I IFN 
PLoS ONE  2014;9(5):e95992.
Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by “in vitro virus” selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.
PMCID: PMC4006813  PMID: 24788966
17.  Regulations of Gene Expression in Medullary Thymic Epithelial Cells Required for Preventing the Onset of Autoimmune Diseases 
Elimination of potential self-reactive T cells in the thymus is crucial for preventing the onset of autoimmune diseases. Epithelial cell subsets localized in thymic medulla [medullary thymic epithelial cells (mTECs)] contribute to this process by supplying a wide range of self-antigens that are otherwise expressed in a tissue-specific manner (TSAs). Expression of some TSAs in mTECs is controlled by the autoimmune regulator (AIRE) protein, of which dysfunctional mutations are the causative factor of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). In addition to the elimination of self-reactive T cells, recent studies indicated roles of mTECs in the development of Foxp3-positive regulatory T cells, which suppress autoimmunity and excess immune reactions in peripheral tissues. The TNF family cytokines, RANK ligand, CD40 ligand, and lymphotoxin were found to promote the differentiation of AIRE- and TSA-expressing mTECs. Furthermore, activation of NF-κB is essential for mTEC differentiation. In this mini-review, we focus on molecular mechanisms that regulate induction of AIRE and TSA expression and discuss possible contributions of these mechanisms to prevent the onset of autoimmune diseases.
PMCID: PMC3752772  PMID: 23986760
medullary thymic epithelial cells; autoimmune disease; NF-κB; TNF receptor family; gene expression
18.  DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells 
BMC Immunology  2012;13:58.
Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.
We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.
These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.
PMCID: PMC3546423  PMID: 23116172
Medullary thymic epithelial cells; Aire; T-DMR
19.  RANKL-RANK interaction in immune regulatory systems 
World Journal of Orthopedics  2012;3(9):142-150.
The interaction between the receptor activator of NF-κB ligand (RANKL) and its receptor RANK plays a critical role in the development and function of diverse tissues. This review summarizes the studies regarding the functions of RANKL signaling in immune regulatory systems. Previous in vitro and in vivo studies have indicated that the RANKL signal promotes the survival of dendritic cells (DCs), thereby activating the immune response. In addition, RANKL signaling to DCs in the body surface barriers controls self-tolerance and oral-tolerance through regulatory T cell functions. In addition to regulating DC functions, the RANKL and RANK interaction is critical for the development and organization of several lymphoid organs. The RANKL signal initiates the formation of clusters of lymphoid tissue inducer cells, which is crucial for lymph node organogenesis. Moreover, the RANKL-RANK interaction controls the differentiation of M cells, specialized epithelial cells in mucosal tissues, that take up and transcytose antigen particles to control the immune response to pathogens or commensal bacterium. The development of epithelial cells localized in the thymic medulla (mTECs) is also regulated by the RANKL-RANK signal. Given that the unique property of mTECs to express a wide variety of tissue-specific self-antigens is critical for the elimination of self-antigen reactive T cells in the thymus, the RANKL-RANK interaction contributes to the suppression of autoimmunity. Future studies on the roles of the RANKL-RANK system in immune regulatory functions would be informative for the development and application of inhibitors of RANKL signaling for disease treatment.
PMCID: PMC3502610  PMID: 23173110
RANKL; T cells; Dendritic cells; Thymus; Medullary thymic epithelial cells; Lymphoid tissue inducer cells; Lymph node; M cells; Peyer’s patches
20.  TNF receptor family signaling in the development and functions of medullary thymic epithelial cells 
Thymic epithelial cells (TECs) provide the microenvironment required for the development of T cells in the thymus. A unique property of medullary thymic epithelial cells (mTECs) is their expression of a wide range of tissue-restricted self-antigens, critically regulated by the nuclear protein AIRE, which contributes to the selection of the self-tolerant T cell repertoire, thereby suppressing the onset of autoimmune diseases. The TNF receptor family (TNFRF) protein receptor activator of NF-κB (RANK), CD40 and lymphotoxin β receptor (LtβR) regulate the development and functions of mTECs. The engagement of these receptors with their specific ligands results in the activation of the NF-κB family of transcription factors. Two NF-κB activation pathways, the classical and non-classical pathways, promote the development of mature mTECs induced by these receptors. Consistently, TNF receptor-associated factor (TRAF6), the signal transducer of the classical pathway, and NF-κB inducing kinase (NIK), the signal transducer of the non-classical pathway, are essential for the development of mature mTECs. This review summarizes the current understanding of how the signaling by the TNF receptor family controls the development and functions of mTEC.
PMCID: PMC3432834  PMID: 22969770
medullary thymic epithelial cells; TNF receptor family; NF-κB; signal transduction; self-tolerance; autoimmune disease
21.  Evolution of the apomixis transmitting chromosome in Pennisetum 
Apomixis is an intriguing trait in plants that results in maternal clones through seed reproduction. Apomixis is an elusive, but potentially revolutionary, trait for plant breeding and hybrid seed production. Recent studies arguing that apomicts are not evolutionary dead ends have generated further interest in the evolution of asexual flowering plants.
In the present study, we investigate karyotypic variation in a single chromosome responsible for transmitting apomixis, the Apospory-Specific Genomic Region carrier chromosome, in relation to species phylogeny in the genera Pennisetum and Cenchrus. A 1 kb region from the 3' end of the ndhF gene and a 900 bp region from trnL-F were sequenced from 12 apomictic and eight sexual species in the genus Pennisetum and allied genus Cenchrus. An 800 bp region from the Apospory-Specific Genomic Region also was sequenced from the 12 apomicts. Molecular cytological analysis was conducted in sixteen Pennisetum and two Cenchrus species. Our results indicate that the Apospory-Specific Genomic Region is shared by all apomictic species while it is absent from all sexual species or cytotypes. Contrary to our previous observations in Pennisetum squamulatum and Cenchrus ciliaris, retrotransposon sequences of the Opie-2-like family were not closely associated with the Apospory-Specific Genomic Region in all apomictic species, suggesting that they may have been accumulated after the Apospory-Specific Genomic Region originated.
Given that phylogenetic analysis merged Cenchrus and newly investigated Pennisetum species into a single clade containing a terminal cluster of Cenchrus apomicts, the presumed monophyletic origin of Cenchrus is supported. The Apospory-Specific Genomic Region likely preceded speciation in Cenchrus and its lateral transfer through hybridization and subsequent chromosome repatterning may have contributed to further speciation in the two genera.
PMCID: PMC3198970  PMID: 21975191
22.  A Pair of Circularly Permutated PDZ Domains Control RseP, the S2P Family Intramembrane Protease of Escherichia coli*S⃞ 
The Journal of Biological Chemistry  2008;283(50):35042-35052.
The σE pathway of extracytoplasmic stress responses in Escherichia coli is activated through sequential cleavages of the anti-σE protein, RseA, by membrane proteases DegS and RseP. Without the first cleavage by DegS, RseP is unable to cleave full-length RseA. We previously showed that a PDZ-like domain in the RseP periplasmic region is essential for this negative regulation of RseP. We now isolated additional deregulated RseP mutants. Many of the mutations affected a periplasmic region that is N-terminal to the previously defined PDZ domain. We expressed these regions and determined their crystal structures. Consistent with a recent prediction, our results indicate that RseP has tandem, circularly permutated PDZ domains (PDZ-N and PDZ-C). Strikingly, almost all the strong mutations have been mapped around the ligand binding cleft region in PDZ-N. These results together with those of an in vitro reaction reproducing the two-step RseA cleavage suggest that the proteolytic function of RseP is controlled by ligand binding to PDZ-N.
PMCID: PMC3259892  PMID: 18945679
23.  Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase* 
The Journal of Biological Chemistry  2016;291(46):23952-23964.
In the mammalian endoplasmic reticulum, oxidoreductin-1α (Ero1α) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O2 to H2O2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H2O2 generation cause cell death, Ero1α activity must be properly regulated. In addition to the four catalytic cysteines (Cys94, Cys99, Cys104, and Cys131) that are located in the flexible active site region, the Cys208–Cys241 pair located at the base of another flexible loop is necessary for Ero1α regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys208–Cys241 disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys208 and Cys241 did not grossly affect the thermal stability or overall shape of Ero1α, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys208 and Cys241 provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1α through reduction of the Cys208–Cys241 disulfide. We propose a mechanism of dual Ero1α regulation by dynamic redox interactions between PDI and the two Ero1α flexible loops that harbor the regulatory cysteines.
PMCID: PMC5104921  PMID: 27703014
disulfide; endoplasmic reticulum (ER); hydrogen peroxide; protein-disulfide isomerase; redox regulation
24.  Mouse model of imiquimod-induced psoriatic itch 
Pain  2016;157(11):2536-2543.
Itch is a major indicator of psoriasis, but the underlying mechanisms behind this symptom are largely unknown. To investigate the neuronal mechanisms of psoriatic itch, we tested whether mice subjected to the imiquimod-induced psoriasis model exhibit itch-associated behaviors. Mice received daily topical applications of imiquimod to the rostral back skin for seven days. Imiquimod-treated mice exhibited a significant increase in spontaneous scratching behavior directed to the treated area as well as touch-evoked scratching (alloknesis). To characterize this model, we measured the mRNA expression levels of pruritogens and itch-relevant receptors/channels using real-time RT-PCR. The mRNA expression of MrgprA3, MrgprC11, and MrgprD decreased gradually over time in the dorsal root ganglion (DRG) cells. There was no significant change in the mRNA expression of TRPV1 or TRPA1 in DRG cells. TRPV4 mRNA expression was transiently increased in the DRG cells, while TRPM8 mRNA was significantly decreased. The mRNA expression levels of histidine decarboxylase and tryptophan hydroxylase 1, as well as the intensity of histamine and serotonin immunoreactivity, were transiently increased in the skin on day 2, returning to baseline by day 7. Histamine H1 receptor (H1R) antagonists, chlorpheniramine and olopatadine, significantly inhibited spontaneous scratching on day 2, but not day 7. Neither chlorpheniramine nor olopatadine affected alloknesis on day 2 or day 7. These results may reflect the limited antipruritic effects of H1R antagonists on human psoriasis. The imiquimod-induced psoriasis model appears to be useful for the investigation of itch and its sensitization in psoriasis.
PMCID: PMC5069152  PMID: 27437787
Psoriasis; chronic itch; histamine; alloknesis; scratching
25.  Aglycon diversity of brain sterylglucosides: structure determination of cholesteryl- and sitosterylglucoside[S] 
Journal of Lipid Research  2016;57(11):2061-2072.
To date, sterylglucosides have been reported to be present in various fungi, plants, and animals. In bacteria, such as Helicobacter pylori, proton NMR spectral analysis of isolated 1-O-cholesteryl-β-d-glucopyranoside (GlcChol) demonstrated the presence of an α-glucosidic linkage. By contrast, in animals, no detailed structural analysis of GlcChol has been reported, in part because animal-derived samples contain a high abundance of glucosylceramides (GlcCers)/galactosylceramides, which exhibit highly similar chromatographic behavior to GlcChol. A key step in vertebrate GlcChol biosynthesis is the transglucosylation reaction catalyzed by glucocerebrosidase (GBA)1 or GBA2, utilizing GlcCer as a glucose donor. These steps are expected to produce a β-glucosidic linkage. Impaired GBA1 and GBA2 function is associated with neurological disorders, such as cerebellar ataxia, spastic paraplegia, and Parkinson’s disease. Utilizing a novel three-step chromatographic procedure, we prepared highly enriched GlcChol from embryonic chicken brain, allowing complete structural confirmation of the β-glucosidic linkage by 1H-NMR analysis. Unexpectedly, during purification, two additional sterylglucoside fractions were isolated. NMR and GC/MS analyses confirmed that the plant-type sitosterylglucoside in vertebrate brain is present throughout embryonic development. The aglycon structure of the remaining sterylglucoside (GSX-2) remains elusive due to its low abundance. Together, our results uncovered unexpected aglycon heterogeneity of sterylglucosides in vertebrate brain.
PMCID: PMC5087873  PMID: 27697915
brain lipids; cholesterol; glycolipids; mass spectrometry; sterols, glucosylceramide; matrix-assisted laser desorption/ionization-tandem mass spectrometry

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