Publications by authors named "Morihiko Nakamura"

20 Publications

  • Page 1 of 1

Quercetin and HSC70 coregulate the anti-inflammatory action of the ubiquitin-like protein MNSFβ.

Mol Biol Rep 2021 Nov 12. Epub 2021 Nov 12.

The Department of Cooperative Medical Research, Office for Regional Collaboration and Innovation, Shimane University, Izumo, 693-8501, Japan.

Background: Quercetin is a flavonol that modifies many cellular processes. Monoclonal nonspecific suppressor factor β is a member of the ubiquitin-like family of proteins that are involved in various biological processes. It has been demonstrated that quercetin regulates the effect of MNSFβ on tumor necrosis factor-α secretion in lipopolysaccharide (LPS)-stimulated macrophages. This study found that quercetin and the heat shock protein HSC70 coregulate the action of MNSFβ.

Methods And Results: Quercetin dose-dependently suppressed the LPS/interferon γ-induced nitric oxide production without cytotoxicity in the macrophage-like cell line Raw264.7. SiRNA knockdown experiments showed that quercetin inhibited the MNSFβ and HSC70 siRNA-mediated enhancement of TNFα and the production of RANTES, a member of C-C chemokine superfamily, in LPS-stimulated Raw264.7 cells. Western blot analysis showed that quercetin and HSC70 regulated ERK1/2 activation and LPS-stimulated IκBα degradation by affecting the complex formation of MNSFβ and the proapoptotic protein Bcl-G. Moreover, MNSFβ is implicated in TLR4/MyD88 signaling but not in TLR3 signaling.

Conclusions: HSC70 is an important chaperone that facilitates the stabilization of MNSFβ. Quercetin may negatively control the function of MNSFβ by regulating the action of the molecular chaperone HSC70. MNSFβ mediates TLR4/Myd88 signaling but not TLR3 signaling.
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http://dx.doi.org/10.1007/s11033-021-06949-yDOI Listing
November 2021

Heat shock protein 60 negatively regulates the biological functions of ubiquitin-like protein MNSFβ in macrophages.

Mol Cell Biochem 2019 Jun 1;456(1-2):29-39. Epub 2019 Feb 1.

Division of Regional Collaborative Medical Research, Office for Regional Collaboration and Innovation, Shimane University, Izumo, 693-8501, Japan.

Monoclonal nonspecific suppressor factor β (MNSFβ) is a ubiquitously expressed ubiquitin-like protein known to be involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently modify its target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 65 kDa MNSFβ adduct from mouse liver lysates by sequential chromatography on DEAE and glutathione S-transferase (GST)-fusioned MNSFβ immobilized on glutathione-Sepharose beads in the presence of ATP. MALDI-TOF mass spectrometry fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and heat shock protein 60 (HSP60), a mitochondrial protein involved in protein folding. Fingerprinting analysis of the MNSFβ adduct demonstrates that MNSFβ conjugates to HSP60 with a linkage between the C-terminal Gly74 and Lys481. HSP60 siRNA neutralized the inhibition of apoptosis by MNSFβ siRNA in LPS/IFNγ-stimulated Raw264.7, a murine macrophage cell line. HSP60 siRNA also down-regulated the enhancement of TNFα production by MNSFβ siRNA in LPS-stimulated Raw264.7 cells. Here, we firstly report that MNSFβ activity is negatively regulated by molecular chaperone.
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http://dx.doi.org/10.1007/s11010-018-3487-5DOI Listing
June 2019

Subchronic intravenous toxicity study of biofunctional ZnO and its application as a fluorescence probe for cell-specific targeting.

J Biochem Mol Toxicol 2019 Apr 31;33(4):e22276. Epub 2018 Dec 31.

Division of Regional Collaborative Medical Research, Office for Regional Collaboration and Innovation, Shimane University.

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early cancer is a great challenge. Herein, we choose the visible-light emitting zinc oxide non-core/shell type nanoparticle (NP) fluorophores (ZHIE) as prototypical materials. We have reported on these materials previously. The results showed that the ZHIE NPs exhibited good water solubility and good biocompatibility. This study was conducted to investigate the toxicity of ZHIE NPs when intravenously administered to mice repeatedly at the dose required for successful tumor imaging in vivo. Anti-macrophage-1 antigen (Mac1), a macrophage differentiation antigen, antibody-conjugated ZHIE NPs successfully realized targeted imaging of murine macrophage cell line Raw264.7 cells. In conclusion, ZHIE NPs are not toxic in vivo and antibody-conjugated ZHIE NPs have great potential in applications, such as single cell labeling.
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http://dx.doi.org/10.1002/jbt.22276DOI Listing
April 2019

Ubiquitin-like protein MNSFβ noncovalently binds to molecular chaperone HSPA8 and regulates osteoclastogenesis.

Mol Cell Biochem 2016 Oct 1;421(1-2):149-56. Epub 2016 Sep 1.

The Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, 693-8501, Japan.

MNSFβ, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFβ-conjugating enzyme(s), we identified a novel target protein for MNSFβ. MALDI-TOF MS fingerprinting revealed that the MNSFβ-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFβ noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFβ and HSPA8 strongly inhibited RANKL-induced osteoclastogenesis from Raw264.7 macrophage-like cells. The same treatment inhibited RANKL-induced ERK1/2 and p38 phosphorylation and TNFα production, suggesting that the association of MNSFβ with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFβ binds to a protein substrate via the noncovalent association and exerts biological effects.
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http://dx.doi.org/10.1007/s11010-016-2795-xDOI Listing
October 2016

Ubiquitin-like protein MNSFβ covalently binds to cytosolic 10-formyltetrahydrofolate dehydrogenase and regulates thymocyte function.

Biochem Biophys Res Commun 2015 Sep 17;464(4):1096-1100. Epub 2015 Jul 17.

The Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo 693-8501, Japan.

MNSFβ is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFβ adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFβ IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFβ preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFβ adduct demonstrate that MNSFβ conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFβ/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFβ/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFβ and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFβ/FDH complex formation may positively regulate apoptosis in thymocytes.
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http://dx.doi.org/10.1016/j.bbrc.2015.07.083DOI Listing
September 2015

Ubiquitin-like protein MNSFβ negatively regulates T cell function and survival.

Immunol Invest 2015 2;44(1):1-12. Epub 2014 Sep 2.

Department of Cooperative Medical Research, Collaboration Center, Shimane University , Izumo , Japan.

Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates apoptosis in macrophages. In this study, we demonstrate that MNSFβ negatively regulates T cell function. In murine T-helper type 2 clone, D10.G4.1 (D10) cells transfected with MNSFβ cDNA, CD3/CD28-induced ERK1/2 phosphorylation leading to IL-4 production was significantly inhibited. The formation of MNSFβ-Bcl-G complex was induced by the CD3/CD28 stimulation. Co-transfection with MNSFβ and Bcl-G greatly enhanced CD3/CD28-induced apoptosis in D10 cells. Similarly, co-over-expression of MNSFβ and Bcl-G caused a marked enhancement of apoptosis in purified splenic T cells. Interestingly, this MNSFβ adduct was also induced in T cells derived from DO11.10 mice stimulated with antigen. Collectively, CD3/CD28-inducible MNSFβ-Bcl-G complex may be involved in the regulation of T cell function and survival.
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http://dx.doi.org/10.3109/08820139.2014.909454DOI Listing
August 2015

Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid.

Sci Technol Adv Mater 2013 Apr 10;14(2):025002. Epub 2013 Apr 10.

Emeritus Researcher, National Institute for Materials Science (NIMS), Tsukuba, Iabaraki 305-0044, Japan.

Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS) with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-)). For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR)) with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR) although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR) both in air and in PBS(-). A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR) in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR). The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.
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http://dx.doi.org/10.1088/1468-6996/14/2/025002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074373PMC
April 2013

Ubiquitin-like protein MNSFβ covalently binds to Bcl-G and enhances lipopolysaccharide/interferon γ-induced apoptosis in macrophages.

FEBS J 2013 Mar 11;280(5):1281-93. Epub 2013 Feb 11.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, Japan.

Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade in the mouse macrophage cell line Raw264.7. In this study, we demonstrate that MNSFβ promotes lipopolysaccharide (LPS)/interferon γ (IFNγ)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFβ small interfering RNA (siRNA), LPS/IFNγ- or NO donor S-nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFβ did not affect inducible nitric-oxide synthase (iNOS) expression in LPS/IFNγ-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFβ and Bcl-G greatly enhanced LPS/IFNγ- induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox-2 activity. Unlike co-transfection with wild-type MNSFβ, co-transfection of a mutant MNSFβ (G74A) and Bcl-G did not result in enhancement of LPS/IFNγ-induced apoptosis. Co-over-expression of MNSFβ and Bcl-G reduced S-nitrosoglutathione-induced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFβ down-regulates the ERK/activator protein 1 (AP-1) signaling cascade which leads to Cox-2 activation. We also observed that MNSFβ-Bcl-G promotes LPS/IFNγ-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox-2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFβ-Bcl-G is due in part to down-regulation of Cox-2 activation in macrophages.
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http://dx.doi.org/10.1111/febs.12120DOI Listing
March 2013

Ubiquitin-like protein MNSFβ regulates TLR-2-mediated signal transduction.

Mol Cell Biochem 2012 May 25;364(1-2):39-43. Epub 2012 Jan 25.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo 693-8501, Japan.

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFβ also covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits the signal pathway upstream of IKK activation, but not downstream of TLR-2 signaling. In this study, we further examined the mechanism of action of MNSFβ in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFβ siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFβ cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFβ-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFβ-adduct was not induced by LPS, indicative of the specificity of TLR-2-mediated signal transduction. Similar observations were seen in BALB/c peritoneal macrophages. Interestingly, 40-kDa MNSFβ-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFβ-adducts may regulate TLR-2 signaling pathway in macrophages.
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http://dx.doi.org/10.1007/s11010-011-1202-xDOI Listing
May 2012

Chronic administration of cardanol (ginkgol) extracted from ginkgo biloba leaves and cashew nutshell liquid improves working memory-related learning in rats.

Biol Pharm Bull 2012 ;35(1):127-9

Showa Pharmaceutical University, Higashi-tamagawagakuen, Machida, Tokyo 194–8543, Japan.

Cardanol (ginkgol) extracted from Ginkgo biloba leaves and cashew nutshell liquid enhances the growth of NSC-34 immortalized motor neuron-like cells and, when chronically administered to young rats, improves working memory-related learning ability as assessed by eight-arm radial maze tasks. These findings suggest that cardanol is one of the components in Ginkgo biloba leaves that improves cognitive learning ability.
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http://dx.doi.org/10.1248/bpb.35.127DOI Listing
June 2012

Anti-allergic effect of the flavonoid myricitrin from Myrica rubra leaf extracts in vitro and in vivo.

Nat Prod Res 2011 Feb;25(4):374-80

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, Japan.

Flavonoids are ingested by the general population as anti-oxidant and anti-inflammatory agents. In this study, we investigated the effects of myricitrin, a flavonoid rich in Myrica rubra leaf, upon anti-inflammatory action. Myrica rubra leaf extracts inhibited pro-inflammatory TNFα production in a macrophage cell line, Raw264.7 cells. We observed that the serum IgE levels in the leaf extract-treated DO11.10, a mouse allergy model, were down-regulated. HPLC was performed to demonstrate that M. rubra leaf extracts contain a large amount of myricitrin. We observed an inhibitory effect of HPLC-purified myricitrin on TNFα production in Raw264.7 cells. Thus, myricitrin may be of potential interest in the management of inflammatory conditions.
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http://dx.doi.org/10.1080/14786411003774320DOI Listing
February 2011

Ubiquitin-like protein MNSFβ/endophilin II complex regulates Dectin-1-mediated phagocytosis and inflammatory responses in macrophages.

Biochem Biophys Res Commun 2010 Oct 16;401(2):257-61. Epub 2010 Sep 16.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo 693-8501, Japan.

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been implicated in the regulation of a variety of cellular events. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G in a macrophage cell line, Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. Most recently, we found that MNSFβ covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits phagocytosis by macrophages. In this study, we further examined the mechanism of action of MNSFβ/endophilin II complex in the phagocytosis of zymosan. MNSFβ/endophilin II I mediated inhibition of phagocytosis in Raw264.7 cells was neutralized by anti-Decti-1, β-glucan receptor, mAb, indicating that MNSFβ/endophilin II is a mediator of Dectin-1 signaling in regulating phagocytosis. The β-glucan-dependent TNFα response to zymosan was significantly increased by the treatment with endophilin II siRNA and/or MNSFβ siRNA. Conversely, cotransfection of endophilin II and MNSFβ cDNAs inhibited the enhancement of zymosan-induced TNFα production. Interestingly, endophilin II siRNA did not affect Pam3CSK4 (TLR2 specific ligand)-induced TNFα production. Endophilin II and/or MNSFβ siRNA enhanced zymosan-induced IκBα degradation. Together, these results demonstrate that MNSFβ/endophilin II inhibits the signal pathway upstream of IKK activation, but not downstream of TLR2 signaling.
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http://dx.doi.org/10.1016/j.bbrc.2010.09.045DOI Listing
October 2010

The ubiquitin-like protein monoclonal nonspecific suppressor factor beta conjugates to endophilin II and regulates phagocytosis.

FEBS J 2009 Nov 1;276(21):6355-63. Epub 2009 Oct 1.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo 693-8501, Japan.

Monoclonal nonspecific suppressor factor beta (MNSFbeta) is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to the intracellular proapoptotic protein Bcl-G in cells of the macrophage cell line Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. In this study, we purified a 62 kDa MNSFbeta adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFbeta IgG-conjugated Sepharose. MALDI-TOF MS fingerprinting revealed that this MNSFbeta adduct consists of an 8.5 kDa MNSFbeta and endophilin II, a member of the endophilin A family. MNSFbeta may conjugate to endophilin II with a linkage between the C-terminal Gly74 and Lys294. We confirmed this result by immunoprecipitation/western blot studies. Endophilin II was ubiquitously expressed in various tissues, although a truncated form was observed in liver. The 62 kDa MNSFbeta-endophilin II was specifically expressed in liver and macrophages. Small interfering RNA-mediated knockdown of endophilin II and/or MNSFbeta promoted phagocytosis of zymosan in Raw264.7 cells. Conversely, cotransfection of endophilin II and MNSFbeta cDNAs inhibited the phagocytosis of zymosan. Such inhibition was not observed in cells expressing a mutant of endophilin II in which Lys294 was replaced by arginine. These results suggest that the post-translational modification of endophilin II by MNSFbeta might be implicated in phagocytosis by macrophages.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07348.xDOI Listing
November 2009

Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages.

Biosci Biotechnol Biochem 2008 Jul 7;72(7):1915-20. Epub 2008 Jul 7.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, Japan.

Monoclonal non-specific suppressor factor beta (MNSFbeta) is a member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta regulates the ERK1/2-MAPK cascade in the macrophage cell line Raw 264.7. In this study, we found evidence that the flavonol quercetin regulates the effect of MNSFbeta on TNFalpha production in LPS-stimulated Raw264.7 cells. Quercetin inhibited MNSFbeta siRNA-mediated enhancement of both TNFalpha production and ERK1/2 phosphorylation in LPS-stimulated Raw264.7 cells. Quercetin decreased the expression of 33.5-kDa MNSFbeta adduct, which is important to the regulation of ERK1/2 activity, in unstimulated Raw264.7 cells. The various flavonoids tested, including other flavonols, did not affect the formation of this adduct. Collectively, MNSFbeta and quercetin might share a common pathway in regulating the ERK1/2 pathway in macrophages. This is the first report describing the involvement of flavonoids in the action of ubiquitin-like proteins.
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http://dx.doi.org/10.1271/bbb.80167DOI Listing
July 2008

The ubiquitin-like protein MNSFbeta regulates ERK-MAPK cascade.

J Biol Chem 2006 Jun 18;281(25):16861-16869. Epub 2006 Apr 18.

Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo 693-8501, Japan; Department of Pediatrics, Faculty of Medicine, Shimane University, Izumo 693-8501, Japan.

MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.
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http://dx.doi.org/10.1074/jbc.M509907200DOI Listing
June 2006

Noncovalent interaction of MNSFbeta, a ubiquitin-like protein, with histone 2A.

Comp Biochem Physiol B Biochem Mol Biol 2005 Feb;140(2):207-10

Cooperative Medical Research Center, Shimane University, 89-1 Enya-cho, Izumo 693-8501, Japan.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSFbeta, an isoform of the MNSF, has been isolated and characterized. MNSFbeta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Most recently, we observed that Ubi-L covalently conjugates to Bcl-G, a novel pro-apoptotic protein. In this study, we observed that Ubi-L noncovalently and specifically binds to histone 2A. The maximum binding was observed at a molar ratio equal to 1 for GST-Ubi-L and 2 for histone 2A. Ubi-L formed complex with histone 2A in the presence of 1% Triton X-100. Free Ubi-L was detected in nuclei from unstimulated murine helper T cell line, D10. The increased amounts of free Ubi-L and some Ubi-L adducts were observed in nuclei from mitogen-activated D10 cells. Interestingly, two Ubi-L adducts were unique to the chromatin fraction of nuclei from the activated D10 cells.
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http://dx.doi.org/10.1016/j.cbpc.2004.09.030DOI Listing
February 2005

Ubiquitin-like polypeptide inhibits cAMP-induced p38 MAPK activation in Th2 cells.

Immunobiology 2004 ;208(5):439-44

Cooperative Medical Research Center, Shimane Medical University, School of Medicine, Izume, Japan.

Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L exhibits an antigen-nonspecific immunosuppressive function on various target cells including murine T helper type 2 (Th2) clone, D10 cells. Ubi-L specifically binds to cell surface receptors on D10 cells. In this study, we observed that Ubi-L inhibited cAMP-induced IL-5 mRNA expression in D10 cells but not in thymoma cell line EL4. In addition, Ubi-L effectively inhibited cAMP-induced p38 MAPK activation in D10 cells. Ubi-L also showed inhibitory activity on IL-5 and IL-13 production by D10 cells stimulated with phorbol ester plus dibutyryl cAMP. Furthermore, Ubi-L inhibited IL-4 production in Th2 cells derived from primary CD4+ T cells.
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http://dx.doi.org/10.1078/0171-2985-00291DOI Listing
November 2004

Grazing exit electron probe microanalysis of submicrometer inclusions in metallic materials.

Anal Chem 2003 Aug;75(15):3831-6

Materials Analysis Station, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan.

Grazing exit electron probe microanalysis (GE-EPMA) is a new method of EPMA in which characteristic X-rays emitted from only near-surface regions of a specimen are detected at extremely low exit angles near 0 degrees (the grazing exit condition). This technique requires the analytical objects exist on a flat surface. Therefore, the GE-EPMA analysis has been used only for the analysis of particles or a thin film on a flat substrate so that there were only few applications for practical analysis. As a new application, we have carried out GE-EPMA analysis of approximately 0.2-microm inclusions on stainless steel, which appeared to be a projection on the specimen surface with chemical etching. The GE-EPMA quantitative results were in excellent agreement with those of inclusions that were extracted from the stainless steel and analyzed by EPMA with conventional exit condition (30 degrees). This method could be, therefore, applied to the analysis of the submicrometer inclusion in a wide variety of metallic materials if the inclusion appears to be a projection with chemical etching treatment.
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http://dx.doi.org/10.1021/ac020740lDOI Listing
August 2003

Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family.

Eur J Biochem 2003 Oct;270(20):4052-8

Cooperative Medical Research Center, Shimane Medical University, Japan.

Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFbeta in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells. In this study, we purified a 33.5 kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-(Ubi-L) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon gamma-activated D.10 cells. The 33.5 kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.
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http://dx.doi.org/10.1046/j.1432-1033.2003.03790.xDOI Listing
October 2003

Biochemical analysis of a T cell receptor alpha-like molecule involved in antigen-nonspecific suppression.

Biochim Biophys Acta 2002 Apr;1589(2):196-202

Department of Biochemistry, Shimane Medical University, Izumo, Japan.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) alpha-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCR(alpha)-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCR(alpha) monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRbeta monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56 degrees C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the J(alpha) region of the TCR(alpha), including the highly conserved motif F-G-X-G-T-X-L.
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http://dx.doi.org/10.1016/s0167-4889(02)00172-6DOI Listing
April 2002
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