Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

DOI： 10.3390/biom11040510

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Language：English   Publishing type：Research paper (scientific journal)  

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki = 0.35 μM for CaMKKα, and Ki = 0.2 μM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

DOI： 10.1021/acs.biochem.0c00149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

DOI： 10.1016/j.bbrc.2020.02.056

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SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1-3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of IgV genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1-3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the IgL gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the IgL promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the IgL promoter leads to an increase in IgL transcription, revealing a role of SRSF1-3 in SHM. Thus, SRSF1-3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1-3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo.

DOI： 10.1016/j.molimm.2020.01.005

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DOI： 10.1016/j.molimm.2019.10.002

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BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

DOI： 10.1016/j.bbagen.2018.12.012

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DOI： 10.1007/978-1-4939-9030-6_23

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DOI： 10.1074/jbc.RA118.006226

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Language：English   Publisher：(一社)日本生物物理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)/5-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKK that exhibits higher basal activity (autonomous activity), activation of the CaMKK/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKK appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKK activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKK at multiple residues by CaMKK-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKK activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKK indicated that Thr(144) phosphorylation by activated AMPK converts CaMKK into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKK mutant. CaMKK mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr(144) phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr(144) antibody revealed phosphorylation of Thr(144) in CaMKK in transfected COS-7 cells that was further enhanced by exogenous expression of AMPK. These results indicate that AMPK-mediated feedback phosphorylation of CaMKK regulates the CaMKK/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKK.

DOI： 10.1074/jbc.M117.805085

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, All, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.07.161

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSFI-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSFI-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, over expression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSFI-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C terminus-dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.02.097

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-BLACKWELL  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-BLACKWELL  

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WILEY-BLACKWELL  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ceca.2016.04.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK phosphorylates Thr(172) in the AMPK subunit more efficiently than CaMKK, with a lower K-m (approximate to 2 m) for AMPK, whereas the CaMKI phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK/CaMKK chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK/Ile(322) in CaMKK confer, at least in part, a distinct recognition of AMPK but not of CaMKI.

DOI： 10.1074/jbc.M116.727867

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

To assess the isoform specificity of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKK alpha mutant (Ala292Thr/Leu233Phe) and a CaMKK beta mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wildtype or the STO-609-resistant mutant of CaMKK alpha was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKK beta mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKK beta is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK. isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

DOI： 10.1021/acs.biochem.5b00149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEDERATION AMER SOC EXP BIOL  

Occurrence of a CSF-1 receptor-dependent monocyte differentiation process driven by IL-34, but not CSF-1. With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b(+) monocytic cells (F4/80(+), Gr-1(-), Ly6C(-), I-A/E-/lo, CD11c(-), CD115(+), CXCR4(+), CCR2(+), CX(3)CR1(-)) when cultured with a Lin(-)c-kit(+) population from mouse spleen cells. The developed CD11b(+) cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

DOI： 10.1189/jlb.0613311

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases -including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
Results: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKI alpha (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K-m values of CaMKK isoforms for GTP (400-500 mu M) were significantly higher than those for ATP (similar to 15 mu M), and a 2- to 4-fold decrease in V-max was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of similar to 45 kDa and similar to 35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
Conclusions: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.

DOI： 10.1186/1471-2091-13-27

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

DOI： 10.1073/pnas.1120368109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21-induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE(2). Treatment of B cells with IL-21 plus PGE(2), but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE(2) receptor isoform, EP4, was responsible for IL-21/PGE(2)-induced B cell death. Thus, PGE(2) is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE(2)-induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3-positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE(2)-induced B cell death in GC B cell selection. The Journal of Immunology, 2011, 187: 4210-4218.

DOI： 10.4049/jimmunol.1100934

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with similar to 600-fold lower K(D)) than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.03.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SW Delta C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW Delta C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW Delta C cells, although the protein might be highly susceptible to degradation. In DT40-SW Delta C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW Delta C cells may be useful for constructing AID libraries for efficient screening of mAbs in vitro. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.04.096

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A hypermutating B cell line DT40 is useful for screening antibodies and improving affinity of the selected antibodies in vitro. To perform affinity maturation efficiently, we generated an engineered DT40 line whose immunoglobulin mutation pattern can be transformed from gene conversion into point mutation by conditional suppression of XRCC3 expression. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.09.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We developed a novel in vitro antibody (Ab) generation system using a hypermutating chicken B cell line (DT40-SW). We suppressed the expression of the Pax5 transcription factor by targeted disruption of the gene to increase Ab production in isolated clones and produce the desired Abs. This single genetic manipulation resulted in a significant enhancement of Ab production without significantly affecting maximum cell density. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2008.09.014

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Language：Japanese   Publisher：PHARMACEUTICAL SOC JAPAN  

Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. The generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and sometimes mAbs for molecules conserved between species are difficult to obtain because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been attempting to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during Culture, thereby generating a useful Ab library for screening mAbs. First, we generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Ab generation system using DT40-SW is useful in the following ways: (1) mAbs for various model antigens including antoantigens can be obtained from the DT40-SW Ab library that is free from immunological tolerance; (2) the switching device of the mutation machinery enables fixing desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Ab production. The Ab generation system will be applicable for obtaining valuable Abs such as antitumor Abs.

DOI： 10.1248/yakushi.129.11

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V-H-encoded H chain (VHT) paired with the lambda 1 or lambda 2 L chain. It has been reported that both two VHT/lambda 1 and VHT/lambda 2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only VHT/lambda 2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the VHT. The VHT/lambda 2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than VHT/lambda 1 B cells. Here, to uncover how VHT/lambda 2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of VHT+ GC B cells at a single cell level. VHT/lambda 2 B cells bearing the unmutated VHT gene were found in the GCs more frequently than VHT/lambda 1 and mutated VHT/lambda 2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of VHT/lambda 2 B cells that mutated their VHT genes preferentially expanded, and finally VHT/lambda 2 B cells bearing the T313A mutation occupied VHT+ GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2008.01.002

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The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (VHT) encoded H chain and the lambda 1 or lambda 2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although VHT/lambda 1 and VHT/lambda 2 IgM were equally produced, VHT/lambda 2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of VHT/lambda 2 B cells for pNP was approximately 50-100-fold higher than that of VHT/lambda 1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of VHT/lambda 2 B cells for affinity maturation. VHT/lambda 2 B cells were more responsive to pNP than VHT/lambda 1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the VHT/lambda 2 B cell population was more potent in migration into GCs than the VHT/lambda 1 counterpart. Thus, it is suggested that the higher BCR affinity of VHT/lambda 2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2006.12.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

DOI： 10.1263/jbb.102.478

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, Fc gamma RIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

A nasal formulation of mometasone furoate (MF) is advantageous in avoiding systemic activity characteristic of glucocorticoids when it is applied topically. To confirm anti-allergic effects of this glucocorticoid formulation elaborately, we investigated whether the drug can suppress the production of IgE antibodies and related cytokines. It we showed that IgE production induced in mice immunized via intranasal route was significantly reduced when the mice were administered MF intranasally. Further, MF was effective in inhibiting production of type-2 helper T cell cytokines in vivo and in vitro. These results provide a immunopharmacological basis for clinical efficacy of this drug.

DOI： 10.1080/08923970600928155

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

DOI： 10.1093/nar/gnj013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To understand the mechanism leading to autoantibody production; it is of importance to reveal how self-components that are otherwise inactive as antigens acquire immunogenicity. One possible mechanism is the generation of structurally modified self-proteins in apoptotic or inflamed tissues. The post-translational modification of proteins might give rise to the generation. of new epitopes to which T and B lymphocytes are not rendered tolerant. Among the protein modifications; this review is focussed on the generation and the immunogenicity of self-proteins carrying 3-nitrotyrosine (NT), an inflammation-associated marker. NT proteins are generated in vivo by nitration with peroxynitrite, which is formed from nitric oxide and superoxide that are released from activated inflammatory cells. Interestingly, many anti-DNA Abs from autoimmune mice have been shown cross-reactive with NT. Analysis of the immunogenicity of NT-carrying self-proteins has revealed that they elicit both Immoral and cellular immune responses in mice. Thus, NT containing epitopes created on self-proteins may serve as a trigger to impair or bypass immunological tolerance. (c) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.autrev.2004.11.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies(Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.11.143

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (VHT)encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had similar to2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, VHT/lambda2 B cells significantly predominated over VHT/lambda1 B cells in MZ-(VHT/lambda1:VHT/lambda2 approximate to 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the VHT/(lambda)2 B cells were further augmented by doubling the VHT gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between VHT/lambda1 and VHT/lambda2 IgM mAbs revealed a polyreactive nature of the VHT/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To explore the link between inflammation and autoimmunity, we analyzed the immunogenicity of 3-nitrotyrosine(NT)-bearing self-proteins, an inflammation-associated marker that is formed by nitration of protein tyrosine residues with peroxynitrite generated during inflammation. An interesting feature of NT is its structural similarity to a synthetic hapten, 4-hydroxy-3-nitrophenylacetyl (NP), with which some anti-DNA antibodies (Abs) have been reported to show cross-reactivity. We confirmed that some of anti-DNA monoclonal Abs (mAbs) obtained from MRL/lpr mice also bound NT as well as NP. Based on these findings, we examined whether NIT-bearing autologous IgG (NT-1gG) as a model of NT-self proteins is immunogenic to induce a DNA-cross-reactive anti-NT Ab response in autologous normal mice. Anti-NT IgM and IgG Ab responses were elicited after the third immunization with NT-IgG. concomitant with an increase in anti-single stranded (ss)DNA titer. Interestingly, a part of anti-NT mAbs thus induced showed cross-reactivity with ssDNA. some of which used VH sequences that were highly homologous to those reported in anti-DNA Abs from NZB/WF1 mice. Splenic T cells primed with NT-IgG. but not with unmodified IgG. showed a proliferative response to the inducing antigen. Collectively, NT-IgG is immunogenic in autologous hosts. and can induce anti-NT Abs that are cross-reactive with ssDNA. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2004.07.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid p-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomial 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system. (C) 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1388-1981(03)00002-7

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DOI： 10.2174/1568010033484124

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

The quasi-monoclonal mouse has limited B cell diversity, whose major (similar to80%) B cell Ag receptors are comprised of the knockin V-H 17.2.25 (VHT)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/lambda1 and VHT/lambda2 IgM were equally produced, VHT/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 Of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that VHT/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an,affinity maturation process.

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A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-2478(02)00083-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high-affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)-knockin mouse whose B cell diversity is limited due to the expression of the site-directed transgene. Immunization of the mouse with p-nitrophenylacetyl (pNP)-conjugated chicken gamma-globulin preferentially led to the production of anti-pNP IgG Ab comprised of non-VHT-encoded H chains and lambda chains. lambda(+) IgG constituted a majority of high-affinity Ab to this hapten. RAG-2 mRNA and the recombination signal sequence break of the lambda1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating X, gene rearrangement and the production of lambda(+) high-affinity anti-pNP IgG. These observations were reproduced in RAG-1-deficient mice that were reconstituted with the spleen cells of the knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.

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Language：English   Publisher：Faculty of Engineering, Okayama University  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Although a high level of IgE is produced after primary infection with Nippostrongylus brasiliensis (Nb), most of the IgE antibodies (Abs) are not specific to the worm. Analyses with Western blotting and enzyme-linked immunosorbent assay (ELISA) revealed that the IgE Abs from Nb-infected BALB/c mice did not show reactivity with Nb-derived excretory-secretory proteins (NES) and antigens present in the cell-free extracts of the worm. Monoclonal IEE Abs obtained from the Nb-infected mice were not reactive with these Nb antigen either. To characterize Nb-induced IgE response, we used (QM x C57BL/6)F1 (QBF1) mice that bear the knock-in 17.2.25 VHDJH segment (VHT) encoding a VH region specific to 4-hydroxy-3-nitrophenylacetyl hapten, and express VHT-encoded antigen receptors on 80-85% of their B cells. Consistent with the frequency of VHT-positive B cells, more than 80% of IgE Abs induced in QBF1 B cells that were cultured with LPS plus IL-4 were found to bear VHT-encoded H chains. In contrast, when QBF1 mice were infected with Nb, less than 10% of Nb-induced IgE Abs were found to use VHT. The QBF1-derived IgE did not react with Nb antigens either. Taken together, data suggest that Nb-induced IgE response in mice is not merely the result of polyclonal activation of B cells, but may involve a mechanism that revises Ig genes secondarily. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-2478(01)00216-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Thymosin alpha1 (T alpha1) is an oligopeptide hormone originally isolated from the thymus gland, and has been reported to have stimulating effects on the differentiation of T cells and NK cells. These immunostimulating properties have been considered to be useful for improving immune disorders associated with various diseases including cancer, AIDS and hepatitis. Here, we characterized immunostimulating properties of Tal in experimental immunodeficiency of mice that was induced by the administration of cyclophosphamide (CY). Repeated injection of 30-300 mug/kg/day of Tal after CY-treatment significantly accelerated the restoration of the reduced number of CD4(+)CD8(+) T cells in the thymus. T alpha1 administration was effective in restoring the suppressed activities of helper T cells and cytotoxic T cells in CY-treated mice. Tal also had stimulating effects on reduced activity of lymphokine-activated killer cells in CY-treated mice. These results indicate that T alpha1 is stimulatory for both humoral and cellular immune responses, thus providing the immunological basis for the clinical benefit of this compound.

DOI： 10.1081/IPH-100102569

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Collagen-induced arthritis (CIA) is an excellent model of rheumatoid arthritis (RA) in humans that is induced in DBA/1 mice immunized with bovine type II collagen (CII). Here, we report that the induction of CIA was effectively suppressed by oral administration of AZ-9, a purified polysaccharide with the average molecular weight of approximately 200 kDa that was produced by a soil bacterium, Klebsiella oxytoca. When AZ-9 was administered at 125-250 mg/kg/day orally for 9 consecutive days after immunization with CII followed by its administration every 3 days, resulted in a marked reduction of the incidence and the severity of CIA. The serum level of anti-CII IgG2a and the production of IFN-gamma and IL-12 in the draining lymph node (LN) cells were significantly lower in AZ-9-administered mice than the untreated control. These findings suggest that orally administered AZ-9 suppressed CIA through attenuating a Th1-type response to CII. AZ-9 could be fragmented into smaller molecules (3-4 kDa) without losing its suppressive activity. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0162-3109(00)00247-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes encoding enzymes of the peroxisomal beta-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to n-alkane (UAS(ALK)) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis (CT-T3A). The 29-bp region (from -289 to -261) present upstream of the TATA sequence was sufficient to induce n-alkane-dependent expression of a reporter gene. Besides n-alkane, UAS(ALK)-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UAS(ALK) were constructed and tested for their UAS activity, It was clarified that the important nucleotides for UAS(ALK) activity were located within 10-bp region from -273 to -261 (5'-TCCTGCACAC-3'). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of Saccharomyces cerevisiae. Similar sequences to UAS(ALK) were also found on several peroxisomal enzyme encoding genes of C. tropicalis.

DOI： 10.1128/JB.182.9.2492-2497.2000

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Here, we report the enhancing effects of nitric oxide (NO) on an IgE antibody response in mice. Anti-trinitrophenyl (TNP) IgE production induced in vitro in TNP keyhole Limpet hemocyanin (KLH)-primed spleen cells was inhibited by approximately 70% when an NO synthase (NOS) inhibitor, L-N-G-monomethyl-L-arginine, was added at 10(-7)-10(-6) M to the lymphocyte culture. On the other hand, addition of NO-generating agents to the culture resulted in a marked enhancement of the IgE production. In contrast, anti-TNP IgM and IgG1 responses were affected only marginally when the IEE production was either suppressed or augmented by these agents. NO did not directly augment IgE class switching in normal B cells stimulated with lipopolysaccharide and interleukin (IL)-4. NO-mediated augmentation of the IgE response is considered to be of a physiological significance because administration of aminoguanidine (AG), an inhibitor of inducible NOS, to immunized mice resulted in a preferential suppression of anti-TNP IgE production in vivo. This may be explained by the observation that AG-administration increased interferon-gamma expression without changing that of IL-4 in the immunized mice. Taken together, these observations suggest a pathophysiological role of NO in the development of IEE-mediated allergic diseases. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0162-3109(99)00158-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press  

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrate that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.

DOI： 10.1385/CBB:32:1-3:285

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.

DOI： 10.1016/S1389-1723(99)80143-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by trio pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-TIE, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption, The homozygous ct-t1a Delta/t1b Delta null mutation abolished the activity of acetoacctyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a Delta/t3b Delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on fz-alkanes (C-10 to C-13), All thiolase activities in this yeast disappeared with the ct-t1a Delta/t1b Delta and ct-t3a Delta/t3b Delta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-TIA locus in the ct-t1b Delta null mutant. The truncated acetoacetyl-CoA thiolase vies solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth hy a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly el en to the beta-oxidation of a C-4 substrate butyrate.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immunochemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-Al-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

DOI： 10.1111/j.1432-1033.1997.00748.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NEW YORK ACAD SCIENCES  

DOI： 10.1111/j.1749-6632.1995.tb19922.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Two transcribed genes encoding 3-ketoacyl-CoA thiolase [(EC 2.3.1.16) Thiolase III], one of the peroxisomal thiolase isozymes, of n-alkane-utilizable yeast, Candida tropicalis, were isolated. Restriction maps of these two genes were very similar. Each of the genes had only one open reading frame consisting of 1224 bp corresponding to 408 amino acid residues. The nucleotide sequences of another thiolase isozyme in peroxisomes of C. tropicalis, acetoacetyl-CoA thiolase (Thiolase IA and IB), have been already determined (Kurihara et al., fur. J. Biochem., 210, 999-1005, 1992). The amino acid sequences of these peroxisomal thiolase isozymes (Thiolases I and III) showed 35% homology. The regulation of biosynthesis of these thiolases in C. tropicalis was compared by RNA and Western blotting analyses. The results revealed that the biosynthesis of Thiolase III in C. tropicalis was strongly induced in a medium containing butyrate or alkanes as a carbon source and a peroxisome proliferator, while Thiolase I was constitutively expressed even in a medium with glucose or acetate, although a slight induction was observed at the levels of transcription and translation in the medium containing butyrate or alkanes. Thus, the regulations of biosyntheses of Thiolases I and III were found to be quite different, in spite of the enzymes in the final step of the peroxisomal fatty acid beta-oxidation system.

DOI： 10.1016/0922-338X(94)90356-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

The coexistence of two thiolase isozymes (acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase), essential for the complete degradation of fatty acids, only in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (Kurihara et al., J. Biochem., 106, 474-478, 1989) is unique in eukaryotic cells. As one of the methods of analysis of molecular information from these isozymes, the calculation of the evolutional distance among thiolases from various organisms suggested that yeast peroxisomal thiolase isozymes are important enzymes in examining the molecular evolution of the fatty acid metabolic pathway and the biogenesis of peroxisomes.

DOI： 10.1016/0922-338X(94)90357-3

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Language：English   Publishing type：Research paper (bulletin of university, research institution)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambdaEMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41 898 Da for thiolase IA and 41 930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.

DOI： 10.1111/j.1432-1033.1992.tb17505.x

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/j.jbiotec.2010.09.628

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Applicant：国立大学法人 岡山大学

Application no：特願2005-247074  Date applied：2005.8.29

Announcement no：特開2007-060907  Date announced：2007.3.15

Patent/Registration no：特許第4482693号  Date issued：2010.4.2

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Applicant：国立大学法人 岡山大学

Application no：特願2004-298072  Date applied：2004.10.12

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Application no：特願2004-298072  Date applied：2004.10.12

Announcement no：特開2006-109711  Date announced：2006.4.27

Patent/Registration no：特許第4487068号  Date issued：2010.4.9

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