Publications by authors named "Tsuyoshi Miyake"

30 Publications

  • Page 1 of 1

Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35.

J Virol 2020 07 30;94(16). Epub 2020 Jul 30.

Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.
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http://dx.doi.org/10.1128/JVI.02100-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394894PMC
July 2020

The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab.

Acta Crystallogr D Struct Biol 2018 07 27;74(Pt 7):681-689. Epub 2018 Jun 27.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.
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http://dx.doi.org/10.1107/S2059798318007878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038385PMC
July 2018

Quantitative evaluation of haze formation of koji and progression of internal haze by drying of koji during koji making.

J Biosci Bioeng 2017 Jul 31;124(1):62-70. Epub 2017 Mar 31.

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Kita-ku, Okayama 701-1296, Japan.

The construction of an experimental system that can mimic koji making in the manufacturing setting of a sake brewery is initially required for the quantitative evaluation of mycelia grown on/in koji pellets (haze formation). Koji making with rice was investigated with a solid-state fermentation (SSF) system using a non-airflow box (NAB), which produced uniform conditions in the culture substrate with high reproducibility and allowed for the control of favorable conditions in the substrate during culture. The SSF system using NAB accurately reproduced koji making in a manufacturing setting. To evaluate haze formation during koji making, surfaces and cross sections of koji pellets obtained from koji making tests were observed using a digital microscope. Image analysis was used to distinguish between haze and non-haze sections of koji pellets, enabling the evaluation of haze formation in a batch by measuring the haze rate of a specific number of koji pellets. This method allowed us to obtain continuous and quantitative data on the time course of haze formation. Moreover, drying koji during the late stage of koji making was revealed to cause further penetration of mycelia into koji pellets (internal haze). The koji making test with the SSF system using NAB and quantitative evaluation of haze formation in a batch by image analysis is a useful method for understanding the relations between haze formation and koji making conditions.
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http://dx.doi.org/10.1016/j.jbiosc.2017.02.011DOI Listing
July 2017

Change in enzyme production by gradually drying culture substrate during solid-state fermentation.

J Biosci Bioeng 2015 Jun 5;119(6):674-7. Epub 2014 Dec 5.

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Kita-ku, Okayama 701-1296, Japan.

The influence of drying the culture substrate during solid-state fermentation on enzyme production was investigated using a non-airflow box. The drying caused a significant increase in enzyme production, while the mycelium content decreased slightly. This suggests that changes in the water content in the substrate during culture affect enzyme production in fungi.
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http://dx.doi.org/10.1016/j.jbiosc.2014.11.005DOI Listing
June 2015

Intra-arterial infusion of thrombin: animal experiments.

Minim Invasive Ther Allied Technol 2014 Jan 30;23(1):52-4. Epub 2013 Sep 30.

Kansai Medical University, Department of Radiology , Osaka , Japan.

Purpose: Thrombin inhibits cadherin on vascular endothelial cells, rapidly and reversibly increasing endothelial permeability. The purpose of this study was to evaluate the feasibility of trans-arterial infusion with thrombin.

Material And Methods: Ten rabbits with right thigh tumor were randomly divided into two groups: A thrombin group and a control group. In the thrombin group, a suspension of thrombin (300 IU), cisplatin (3 mg), lipiodol (0.3 ml) and iopamidol (0.3 ml) was infused into the right femoral artery. In the control group, a suspension of cisplatin, lipiodol and iopamidol was infused. Platinum concentrations in plasma were measured five and ten minutes after administration. Platinum concentrations were also measured in tumor specimens excised 30 minutes after infusion.

Results: At both five and ten minutes after infusion, platinum concentrations in plasma were significantly lower for the thrombin group than for the control group. Platinum concentration in tumor tissue was significantly higher for the thrombin group than for the control group.

Conclusion: The present results suggest that transarterial infusion with thrombin may offer a number of pharmacological advantages.
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http://dx.doi.org/10.3109/13645706.2013.831107DOI Listing
January 2014

Rapid enzyme production and mycelial growth in solid-state fermentation using the non-airflow box.

J Biosci Bioeng 2013 Nov 30;116(5):585-90. Epub 2013 May 30.

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Kita-ku, Okayama 701-1296, Japan. Electronic address:

Solid-state fermentation (SSF) has become an attractive alternative to submerged fermentation (SMF) for the production of enzymes, organic acids, and secondary metabolites, while there are many problems during the culture of SSF. We recently created a SSF system using a non-airflow box (NAB) in order to resolve the problems, which enabled the uniform culture in the whole substrate and high yield of many enzymes. In this paper, further characterization of SSF using the NAB was carried out to obtain other advantages. The NAB culture under the fixed environmental condition exhibited a rapid increase in enzyme production at earlier phase during the culture compared with conventional SSF. Total mycelial growth also exhibited the same trend as enzyme production. Thus, the increase in the rate of the enzyme production was thought to mainly be attributed to that of the growth. To support it, it was suggested that the NAB culture resulted in most optimal water activity for the growth just at the log phase. In addition, the NAB culture was able to achieve high reproducibility of enzyme production, derived from uniform condition of the substrate during the culture. The results indicate that the NAB culture has many benefits for SSF.
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http://dx.doi.org/10.1016/j.jbiosc.2013.04.024DOI Listing
November 2013

Uniform culture in solid-state fermentation with fungi and its efficient enzyme production.

J Biosci Bioeng 2011 Mar 16;111(3):300-5. Epub 2010 Dec 16.

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Kita-ku, Okayama 701-1296, Japan.

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.
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http://dx.doi.org/10.1016/j.jbiosc.2010.11.008DOI Listing
March 2011

Mitotic down-regulation of p190RhoGAP is required for the successful completion of cytokinesis.

J Biol Chem 2010 Aug 9;285(35):26923-26932. Epub 2010 Jun 9.

Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908. Electronic address:

p190RhoGAP-A (p190) is a GTPase-activating protein known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of Rho intrinsic GTPase activity. We have previously shown that p190 protein levels are cell cycle-regulated, decreasing in mitosis, and that this decrease is mediated by the ubiquitin-proteasome pathway. In addition, overexpression of p190 results in decreased RhoGTP levels at the cleavage furrow during cytokinesis, p190 and the RhoGEF Ect2 play opposing roles in cytokinesis, and sustained levels of p190 in mitosis are associated with cytokinesis failure, all findings that suggest but do not directly demonstrate that completion of cytokinesis is dependent on reduced levels of p190. Here we report, using an RNAi reconstitution approach with a degradation-resistant mutant, that decreased p190 levels are required for successful cytokinesis. We also show that the multinucleation phenotype is dependent on p190 RhoGAP activity, determine that the N-terminal GBDS1 region is necessary and sufficient for p190 mitotic ubiquitination and degradation, and identify four N-terminal residues as necessary for the degradation of p190 in mitosis. Our data indicate that in addition to activation of RhoGEF(s), reduction of RhoGAP (p190) is a critical mechanism by which increased RhoGTP levels are achieved in late mitosis, thereby ensuring proper cell division.
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http://dx.doi.org/10.1074/jbc.M110.103804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930692PMC
August 2010

Epidermal growth factor receptor translocation to the mitochondria: regulation and effect.

J Biol Chem 2009 Dec 19;284(52):36592-36604. Epub 2009 Oct 19.

Department of Microbiology and the Cancer Center, University of Virginia, Charlottesville, Virginia 22908. Electronic address:

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.
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http://dx.doi.org/10.1074/jbc.M109.000760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794774PMC
December 2009

Development and media regulate alternative splicing of a methyltransferase pre-mRNA in Monascus pilosus.

J Agric Food Chem 2009 May 15;57(10):4162-7. Epub 2009 Apr 15.

South China Botanical Garden, Chinese Academy of Sciences, 723 Xingkelu, Guangzhou 510650, China.

Two alternatively spliced mRNAs (d- and l-MpLaeA) of a methyltransferase gene (MpLaeA) were identified from Monascus pilosus IFO4520 and its mutant MK-1. Alternative splicing of the MpLaeA pre-mRNA occurred in the 5'-untranslated region (5'-UTR). The alternative splicing patterns of MpLaeA were regulated by the fungal growth stage and the principal nutrients: that is, the short l-MpLaeA mRNA was a constitutive transcript at all growth stages and different carbon or nitrogen sources, but the glutamate and NaNO(3) as main nitrogen source could up-regulate the long d-MpLaeA mRNA form. The long spliced 5'-UTR of d-MpLaeA blocked GFP expression in Escherichia coli , suggesting that d-MpLaeA mRNA was an ineffective spliced mRNA. Down-regulation of MpLaeA by transgenic antisense d-MpLaeA cDNA resulted in decreasing synthesis of monacolin K in M. pilosus. This suggested that the alternative splicing of MpLaeA mRNA might regulate the synthesis of monacolin K.
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http://dx.doi.org/10.1021/jf9004109DOI Listing
May 2009

Molecular mechanisms in two cell death-types, necrosis and apoptosis, induced by 5-fluoro-2'-deoxyuridine.

Nucleic Acids Symp Ser (Oxf) 2008 (52):627-8

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.
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http://dx.doi.org/10.1093/nass/nrn317DOI Listing
November 2010

Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine.

Genomics 2008 Jul;92(1):9-17

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.
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http://dx.doi.org/10.1016/j.ygeno.2008.02.002DOI Listing
July 2008

Transcriptional regulation of Saccharomyces cerevisiae CYS3 encoding cystathionine gamma-lyase.

Curr Genet 2008 Apr 4;53(4):225-34. Epub 2008 Mar 4.

Department of Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.

In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine gamma-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (-311 to -303) and a CGC motif (CGCCACAC; -193 to -186), which is one base shorter than the CCG motif, in the 5'-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; -217 to -210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs.
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http://dx.doi.org/10.1007/s00294-008-0181-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668581PMC
April 2008

Cep57, a multidomain protein with unique microtubule and centrosomal localization domains.

Biochem J 2008 Jun;412(2):265-73

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to gamma-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.
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http://dx.doi.org/10.1042/BJ20071501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351815PMC
June 2008

Proteome and transcriptome analysis of cell death induced by 5-fluoro-2'-deoxyuridine.

Nucleic Acids Symp Ser (Oxf) 2007 (51):433-4

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

5-fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. Previously, we have been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. To understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome of F28-7 and F28-7-A strain after treated with FUdR, it was found that 5 proteins were up-regulated and 11 proteins were down-regulated in F28-7-A strain. Furthermore, transcriptome analysis shows that 94 genes were up-regulated and 164 genes were downregulated in F28-7-A strain. Identified proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. Our results suggested that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.
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http://dx.doi.org/10.1093/nass/nrm217DOI Listing
April 2008

Identification of a cerulenin resistance gene from Monascus pilosus.

DNA Seq 2007 Feb;18(1):68-72

South China Botanical Garden, Chinese Academy of Sciences. Guangzhou, P. R. China.

Detoxification is essential for the fungal growth in the drug stress environments, and the multidrug transporters play an important role in this process. Here a cerulenin transporter gene (MpMdt, AB206476) was identified from Monascus pilosus. MpMdt mRNA contains 1951 bp and encodes a protein of 559 amino acid residues with 11 trans-membrane domains; and there is no difference in the sequence of MpMdt mRNA between the wild type M. pilosus IFO4520 and its cerulenin resistant mutant MK-1. Up-expression of MpMdt renders the cerulenin resistance of the mutant MK-1. Over-expression of MpMdt could also increase the cerulenin tolerance in the transgenic M. pilosus IFO4520. These results suggested that MpMdt is able to efflux-transport the anti-fungal antibiotic cerulenin and increase the cerulenin resistance of M. pilosus.
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http://dx.doi.org/10.1080/10425170601108670DOI Listing
February 2007

Epidural spinal myelolipoma in a dog.

J Am Anim Hosp Assoc 2007 Mar-Apr;43(2):132-5

Faculty of Applied Biological Sciences, Department of Clinical Veterinary Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193 Japan.

Epidural spinal myelolipoma was diagnosed in a 13-year-old, male Siberian husky that was referred for evaluation of progressive pelvic limb paresis and urinary incontinence. An epidural mass was detected by magnetic resonance imaging and computed tomography. The mass was removed and identified histopathologically as an epidural myelolipoma. Pelvic limb paresis improved after surgery, but urinary retention associated with neurological bladder dysfunction persisted.
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http://dx.doi.org/10.5326/0430132DOI Listing
July 2008

Proteome and transcriptome analysis of 5-fluoro-2'-deoxyuridine-induced cell death mechanisms.

Nucleic Acids Symp Ser (Oxf) 2006 (50):101-2

Faculty of Pharmaceutical Sciences, Okayama University, Tushima, Okayama 700-8530, Japan.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. The inhibition of thymidylate synthase causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. We have previously been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. In this report, in order to understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome from F28-7 strain and F28-7-A strain, it was found that ten proteins were increased and six proteins were decreased in F28-7-A strain. Furthermore, transcriptome analysis shows that 127 genes were increased and 181 genes were decreased in F28-7-A strain. These differentially expressed proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. These two techniques clarified numerous features in F28-7 strain and F28-7-A strain. Our results revealed that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.
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http://dx.doi.org/10.1093/nass/nrl050DOI Listing
August 2007

Repression of secondary metabolite production by exogenous cAMP in Monascus.

Biosci Biotechnol Biochem 2006 Jun;70(6):1521-3

Industrial Technology Center of Okayama Prefecture, Haga, Okayama.

The cAMP signal pathway controls various biological functions, including secondary metabolism of filamentous fungi. We found that exogenous cAMP represses the production of lovastatin, red pigments, and citrinin in Monascus. Interestingly, a mutant MK-1 with increased lovastatin and red pigments production was not influenced by cAMP on these productions, indicating that cAMP signaling might be lacking in MK-1.
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http://dx.doi.org/10.1271/bbb.50686DOI Listing
June 2006

Effects of the principal nutrients on lovastatin production by Monascus pilosus.

Biosci Biotechnol Biochem 2006 May;70(5):1154-9

Industrial Technology Center of Okayama Prefecture, Japan.

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.
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http://dx.doi.org/10.1271/bbb.70.1154DOI Listing
May 2006

Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels.

J Agric Food Chem 2005 Aug;53(16):6272-5

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.

Nasunin, delphinidin-3-(p-coumaroylrutinoside)-5-glucoside, an antioxidant anthocyanin isolated from eggplant peels, was demonstrated as an angiogenesis inhibitor. Nasunin at higher 10 microM suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring. The effect of nasunin was examined in various in vitro angiogenesis models using human umbilical vein endothelial cells (HUVECs). Nasunin suppressed HUVEC proliferation in a dose-dependent manner (50-200 microM); however, it had no significant effect on HUVEC chemotaxis in a Boyden chamber assay and HUVEC tube formation on a reconstituted basement membrane. These results imply that nasunin with both antioxidant and antiangiogenic activities might be useful to prevent angiogenesis-related diseases.
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http://dx.doi.org/10.1021/jf050796rDOI Listing
August 2005

Light effects on cell development and secondary metabolism in Monascus.

J Ind Microbiol Biotechnol 2005 Mar 5;32(3):103-8. Epub 2005 Mar 5.

Industrial Technology Center of Okayama Prefecture, Haga 5301, Okayama, 701-1296, Japan.

In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms, including filamentous fungi. We have found that both red light and blue light affect development in Monascus, influencing the processes of mycelium and spore formation, and the production of secondary metabolites such as gamma-aminobutyric acid, red pigments, monacolin K and citrinin. Additionally, we observed that the wavelength of light affects these developmental and physiological processes in different ways. These findings suggest that Monascus possesses a system for differential light response and regulation.
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http://dx.doi.org/10.1007/s10295-005-0209-2DOI Listing
March 2005

Functional and physical interactions between autonomously replicating sequence-binding factor 1 and the nuclear transport machinery.

Traffic 2004 Dec;5(12):925-35

Department of Biochemistry and Molecular Genetics, School of Medicine, PO Box 800733, University of Virginia, Charlottesville, VA 22908-0733, USA.

Autonomously replicating sequence-binding factor 1 (Abf1p) is a site-specific DNA binding protein in Saccharomyces cerevisiae that functions to regulate multiple nuclear events including DNA replication, transcriptional activation, and gene silencing. Previous work indicates that the multiple functions of Abf1p are conferred by the carboxy-terminus of the protein, which can be further dissected into two important clusters of amino acid residues (CS1 and CS2). Here we present genetic and cell biological evidence for a critical role of CS1 in proper nuclear localization of Abf1p. Mutations in CS1 cause severe defects in cell growth, nuclear translocation, and Abf1p-mediated gene regulation, which can be rescued by a heterologous nuclear localization sequence (NLS). In addition, the CS1-domain can mediate the import of a CS1-GFP fusion protein. Importantly, the CS1-mediated nuclear import depends on the Ran guanine nucleotide exchange factor Prp20p. Interestingly, a single amino acid change in CS1 (K625I) also causes the protein to be exported out of the nucleus via the Crm1p-dependent pathway. The temperature-sensitive growth phenotype of this particular mutant can be overcome by overexpression of Kap121p/Pse1p, a well-established nuclear transport receptor. Biochemical studies indicate that Pse1p binds to a region of Abf1p upstream of CS1 in a RanGTP-sensitive manner, suggesting that Abf1p has a second distinct NLS and can be imported into the nucleus by several overlapping pathways. We propose that the link between Abf1p and the nuclear transport machinery may also be important for partitioning multiple Abf1p-mediated nuclear processes.
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http://dx.doi.org/10.1111/j.1600-0854.2004.00233.xDOI Listing
December 2004

Comparison of ABF1 and RAP1 in chromatin opening and transactivator potentiation in the budding yeast Saccharomyces cerevisiae.

Mol Cell Biol 2004 Oct;24(20):9152-64

Wadsworth Center, Albany, NY 12201-2002, USA.

Autonomously replicating sequence binding factor 1 (ABF1) and repressor/activator protein 1 (RAP1) from budding yeast are multifunctional, site-specific DNA-binding proteins, with roles in gene activation and repression, replication, and telomere structure and function. Previously we have shown that RAP1 can prevent nucleosome positioning in the vicinity of its binding site and have provided evidence that this ability to create a local region of "open" chromatin contributes to RAP1 function at the HIS4 promoter by facilitating binding and activation by GCN4. Here we examine and directly compare to that of RAP1 the ability of ABF1 to create a region of open chromatin near its binding site and to contribute to activated transcription at the HIS4, ADE5,7, and HIS7 promoters. ABF1 behaves similarly to RAP1 in these assays, but it shows some subtle differences from RAP1 in the character of the open chromatin region near its binding site. Furthermore, although the two factors can similarly enhance activated transcription at the promoters tested, RAP1 binding is continuously required for this enhancement, but ABF1 binding is not. These results indicate that ABF1 and RAP1 achieve functional similarity in part via mechanistically distinct pathways.
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http://dx.doi.org/10.1128/MCB.24.20.9152-9164.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517901PMC
October 2004

Genome-wide analysis of ARS (autonomously replicating sequence) binding factor 1 (Abf1p)-mediated transcriptional regulation in Saccharomyces cerevisiae.

J Biol Chem 2004 Aug 10;279(33):34865-72. Epub 2004 Jun 10.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA 22908-0733, USA.

Autonomously replicating sequence-binding factor-1 (Abf1p) is an essential sequence-specific transcription factor in Saccharomyces cerevisiae that participates in multiple nuclear events including DNA replication, transcription activation, and gene silencing. Numerous gene-specific analyses have implicated Abf1p in the transcriptional control of genes involved in a diverse range of cellular functions, leading to the notion that Abf1p acts as a global transcriptional regulator. Here we report findings from a genome-wide comparison of the gene expression profiles in the wild-type and abf1-1 temperature-sensitive mutant. The study identifies a total of 86 Abf1p-regulated genes (1.4% of the genome) of which 50 are activated and 36 are repressed by Abf1p. Interestingly, Abf1p binds to its own promoter in vivo and strongly represses its own transcription, suggesting a potential negative regulatory loop in Abf1p-mediated gene regulation. A comparison of our microarray data with the available databases reveals a significant overlap of genes regulated by Abf1p and those by several general transcription factors such as Mot1p and TAFs (TATA-binding protein-associated factors). Different mutant alleles of abf1 affect Abf1p-mediated transcription in a gene-dependent manner. Furthermore, Abf1p in vivo is associated with the promoter region of most Abf1p-activated but not with that of most Abf1p-repressed genes. Taken together, these results strongly suggest distinct underlying mechanisms by which Abf1p regulates gene expression.
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http://dx.doi.org/10.1074/jbc.M405156200DOI Listing
August 2004

Acivicin induce filamentous growth of Saccharomyces cerevisiae.

Biosci Biotechnol Biochem 2003 Oct;67(10):2283-5

Industrial Technology Center of Okayama Prefecture, Haga, Okayama, Japan.

The antibiotic acivicin is a known inhibitor of gamma-glutamyl transpeptidase (gammaGTP). We found that acivicin can induce filamentous growth in both diploid and haploid cells of Saccharomyces cerevisiae. This phenomenon is not related to the inhibition of gammaGTP or interference in glutathione metabolism. Interestingly, yeasts used in the brewing industry are more sensitive to acivicin, suggesting that this dimorphological differentiation may be related to some characteristics of these particular strains.
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http://dx.doi.org/10.1271/bbb.67.2283DOI Listing
October 2003

Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

J Biol Chem 2003 Oct 4;278(41):39632-6. Epub 2003 Aug 4.

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama 701-1296, Japan.

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.
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http://dx.doi.org/10.1074/jbc.M302084200DOI Listing
October 2003

Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA box-binding protein (TBP).

Nucleic Acids Res 2003 Feb;31(4):1261-74

Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II. Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure. In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-DeltaTAND) and identified two DeltaTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP. In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene. Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo. Interestingly, a set of genes affected in the taf1-DeltaTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP. Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-DeltaTAND mutant by different mechanisms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150217PMC
http://dx.doi.org/10.1093/nar/gkg180DOI Listing
February 2003

General regulatory factors (GRFs) as genome partitioners.

J Biol Chem 2002 Nov 27;277(44):41736-43. Epub 2002 Aug 27.

CNRS/ENSL 5665, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, France.

Insulators are sequences that uncouple adjacent chromosome domains. Here we have shown that Saccharomyces cerevisiae Rap1p and Abf1p proteins are endowed with a potent insulating capacity. Insulating domains in Rap1p coincide with previously described transcription activation domains, whereas four adjacent subdomains spanning the whole of the Abf1p C terminus (440-731) were found to display autonomous insulating capacity. That both Rap1p and Abf1p silencing domains either contain or largely overlap with an insulating domain suggests that insulation conveys some undefined chromosome organization capacity that also contributes a function in silencing. Together with Reb1p and Tbf1p, previously involved in the activity of Saccharomyces cerevisiae subtelomeric insulators, insulating potential emerges as a supplementary common property of General Regulatory Factors (GRFs). Thus GRFs, which bind to sites scattered throughout the genome within promoters, would not only play a key role in regulating gene expression but also partition the genome in functionally independent domains.
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http://dx.doi.org/10.1074/jbc.M202578200DOI Listing
November 2002

Identification of a multifunctional domain in autonomously replicating sequence-binding factor 1 required for transcriptional activation, DNA replication, and gene silencing.

Mol Cell Biol 2002 Jan;22(2):505-16

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-0733, USA.

Autonomously replicating sequence-binding factor 1 (ABF1) is a multifunctional, site-specific DNA binding protein that is essential for cell viability in Saccharomyces cerevisiae. ABF1 plays a direct role in transcriptional activation, stimulation of DNA replication, and gene silencing at the mating-type loci. Here we demonstrate that all three activities of ABF1 are conferred by the C terminus of the protein (amino acids [aa] 604 to 731). Furthermore, a detailed mutational analysis has revealed two important clusters of amino acid residues in the C terminus (C-terminal sequence 1 [CS1], aa 624 to 628; and CS2, aa 639 to 662). While both regions play a pivotal role in supporting cell viability, they make distinct contributions to ABF1 functions in various nuclear processes. CS1 specifically participates in transcriptional silencing and/or repression in a context-dependent manner, whereas CS2 is universally required for all three functions of ABF1. When tethered to specific regions of the genome, a 30-aa fragment that contains CS2 alone is sufficient for activation of transcription and chromosomal replication. In addition, CS2 is responsible for ABF1-mediated chromatin remodeling. Based on these results, we suggest that ABF1 may function as a chromatin-reorganizing factor to increase accessibility of the local chromatin structure, which in turn facilitates the action of additional factors to establish either an active or repressed chromatin state.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC139751PMC
http://dx.doi.org/10.1128/mcb.22.2.505-516.2002DOI Listing
January 2002