Publications by authors named "Yuki Nakaya"

28 Publications

  • Page 1 of 1

Gas Cell Infrared and Attenuated Total Reflection Infrared Spectroscopic Studies for Organic-Inorganic Interactions in Adsorption of Fulvic Acid on the Goethite Surface Generating Carbon Dioxide.

Appl Spectrosc 2021 Feb 18:3702821991219. Epub 2021 Feb 18.

Department of Earth and Space Science, Graduate School of Science, 13013Osaka University, Toyonaka-shi, Japan.

The generation of carbon dioxide (CO) from Nordic fulvic acid (FA) solution in the presence of goethite (α-FeOOH) was observed in FA-goethite interaction experiments at 25-80 ℃. CO generation processes observed by gas cell infrared (IR) spectroscopy indicated two steps: the zeroth order slower CO generation from FA solution commonly occurring in the heating experiments of the FA in the presence and absence of goethite (activation energy: 16-19 kJ mol), and the first order faster CO generation from FA solution with goethite (activation energy: 14 kJ mol). This CO generation from FA is possibly related to redox reactions between FA and goethite. In situ attenuated total reflection infrared (ATR-IR) spectroscopic measurements indicated rapid increases with time in IR bands due to COOH and COO of FA on the goethite surface. These are considered to be due to adsorption of FA on the goethite surface possibly driven by electrostatic attraction between the positively charged goethite surface and negatively charged deprotonated carboxylates (COO) in FA. Changes in concentration of the FA adsorbed on the goethite surface were well reproduced by the second order reaction model giving an activation energy around 13 kJ mol. This process was faster than the CO generation and was not its rate-determining step. The CO generation from FA solution with goethite is faster than the experimental thermal decoloration of stable structures of Nordic FA in our previous report possibly due to partial degradations of redox-sensitive labile structures in FA.
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http://dx.doi.org/10.1177/0003702821991219DOI Listing
February 2021

Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay.

BMC Infect Dis 2020 Aug 6;20(1):585. Epub 2020 Aug 6.

Sensing System Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.

Background: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation.

Methods: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays.

Results: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R = 0.931, P = 0.000066).

Conclusions: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.
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http://dx.doi.org/10.1186/s12879-020-05317-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407439PMC
August 2020

Single-atom Pt in intermetallics as an ultrastable and selective catalyst for propane dehydrogenation.

Nat Commun 2020 Jun 5;11(1):2838. Epub 2020 Jun 5.

Institute for Catalysis, Hokkaido University, N21, W10, Sapporo, 001-0021, Japan.

Propylene production via propane dehydrogenation (PDH) requires high reaction temperatures to obtain sufficient propylene yields, which results to prominent catalyst deactivation due to coke formation. Developing highly stable catalysts for PDH without deactivation even at high temperatures is of great interest and benefit for industry. Here, we report that single-atom Pt included in thermally stable intermetallic PtGa works as an ultrastable and selective catalyst for PDH at high temperatures. Intermetallic PtGa displays three-hold-Pt ensembles and single Pt atoms isolated by catalytically inert Ga at the surface, the former of which can be selectively blocked and disabled by Pb deposition. The PtGa-Pb/SiO catalyst exhibits 30% conversion with 99.6% propylene selectivity at 600 °C for 96 h without lowering the performance. The single-atom Pt well catalyzes the first and second C-H activation, while effectively inhibits the third one, which minimizes the side reactions to coke and drastically improves the selectivity and stability.
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http://dx.doi.org/10.1038/s41467-020-16693-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275083PMC
June 2020

Three dimensional excitation-emission matrix fluorescence spectroscopy of typical Japanese soil powders.

Spectrochim Acta A Mol Biomol Spectrosc 2020 Jun 21;233:118188. Epub 2020 Feb 21.

HORIBA, Ltd., 2 Miyanohigashi-cho, Kisshoin, Minami-ku, Kyoto 601-8510, Japan.

Front face fluorescence spectroscopy of typical Japanese soil powders (soil A: Typic Hapludand; soil H: Typic Hydraquent; soil Y: Typic Paleudult) has been conducted. Three dimensional excitation-emission matrix fluorescence spectra of the 100 wt% soils showed similar fluorescence patterns to each other. The fluorescence patterns were similar between the soil samples and their residues after extraction by NaOH solution for 60 min. In order to examine fluorescence extinction from a view point of whiteness of the soils, the soil powders were mixed with white and black diluents (AlO and FeO) and fluorescence spectra of the mixtures were measured at 450 nm excitation. At low levels of dilution with AlO (2-100 wt% of A; 50-100 wt% of H and Y), the fluorescence intensities increased with dilution. At high levels of dilution with AlO, the fluorescence intensities decreased with dilution. On the other hand, fluorescent intensities decreased by dilution with FeO. These results suggested inner filter effect-like fluorescence extinction by (1) large amount of blackish organic compounds giving high total carbon value and (2) blackish non-fluorescent mineral compounds. In order to correct the fluorescence intensities of the mixtures containing the sample soils and the diluents, we preliminary applied a correction method based on the Kubelka-Munk theory using diffuse reflectance. The corrected fluorescence intensities of samples with white diluents (AlO) were described by a simple fluorescence response model having saturation values.
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http://dx.doi.org/10.1016/j.saa.2020.118188DOI Listing
June 2020

Corrigendum to "Susceptibility of domestic animals to pseudotype virus bearing RD-114 virus envelope protein" [Gene 567(2) (2015) 189-195].

Gene 2019 03 15;690:137. Epub 2019 Jan 15.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan; Laboratory of Virolution, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto, University, Kyoto, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.gene.2019.01.002DOI Listing
March 2019

Nondestructive Spectroscopic Tracing of Simulated Formation Processes of Humic-Like Substances Based on the Maillard Reaction.

Appl Spectrosc 2018 Aug 29;72(8):1189-1198. Epub 2018 May 29.

2 Department of Bioresource Sciences, Faculty of Agriculture, Ryukoku University, Shiga, Japan.

The formation processes of humic-like substances have been simulated by heating a glycine and ribose mixed solution (0.1 mol L) at 80 ℃ using the Maillard reaction. Ultraviolet-visible (UV-Vis), three-dimensional excitation emission spectroscopy and size exclusion liquid chromatography succeeded in quantitatively tracing increases of the products during the heating of glycine and ribose mixed solution (0.1 mol L). Two-dimensional correlation spectroscopic analyses suggested that a band area around 280 nm ( UV) and 254 nm absorbance ( UV) can be used as measures of the formation of furfural-like intermediates and humic-like products, respectively. They were monitored using in situ UV-Vis spectroscopy with the original heatable liquid cell at 60-80 ℃. Kinetic analyses of the obtained data gave activation energies of 91.4-96.6 kJ mol. These nondestructive measurements by an in situ spectroscopic method did not require any additional procedures including drying or extracting the solution and they can be effectively used for direct tracing of the reaction progress and/or decomposition.
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http://dx.doi.org/10.1177/0003702818775737DOI Listing
August 2018

Efficacy and Patient Satisfaction of the Weekly DPP-4 Inhibitors Trelagliptin and Omarigliptin in 80 Japanese Patients with Type 2 Diabetes.

Intern Med 2017 Oct 6;56(19):2563-2569. Epub 2017 Sep 6.

Division of Diabetes, Department of Internal Medicine, Aichi Medical University School of Medicine, Japan.

Objective We investigated the efficacy, safety, and patient satisfaction of once-weekly DPP-4 inhibitors (DPP-4Is). Methods Either of two once-weekly DPP-4Is, trelagliptin or omarigliptin, was administered alone or in combination with other antidiabetic drugs in 80 outpatients with type 2 diabetes mellitus for 3 months. The HbA1c, glycoalbumin (GA), body weight, and the Diabetes Treatment Satisfaction Questionnaire (DTSQ) scores were evaluated. Results Patients switching from other daily DPP-4Is (n=29) showed no significant changes in the HbA1c or GA levels. However, the HbA1c and GA levels of patients who had been naïve to DPP-4Is (n=37) significantly improved from 9.31±2.53% to 7.02±1.20% (p<0.001) and 26.7±11.8% to 17.3±5.7% (p<0.001), respectively. Several non-serious adverse events were reported, including nausea (n=1), abdominal distension (n=1), and constipation (n=1). In the DTSQs, the total score for six questions on the primary factors representing patient treatment satisfaction was not markedly changed in patients switching from daily to weekly DPP-4Is but was significantly improved from 21.0 to 28.0 (p<0.001) in patients naïve to DPP-4Is. Conclusion These findings suggest that the use of a once-weekly DPP-4I is effective and well-tolerated in diabetes treatment and improves treatment satisfaction.
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http://dx.doi.org/10.2169/internalmedicine.8184-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658520PMC
October 2017

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA.

mBio 2017 07 5;8(4). Epub 2017 Jul 5.

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. Autoimmune diseases like Aicardi-Goutières syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease.
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http://dx.doi.org/10.1128/mBio.00944-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573678PMC
July 2017

Sodium-glucose Co-transporter 2 Inhibitors Reduce the Abdominal Visceral Fat Area and May Influence the Renal Function in Patients with Type 2 Diabetes.

Intern Med 2017 17;56(6):597-604. Epub 2017 Mar 17.

TDE Healthcare Corporation TOSAKI Clinic for Diabetes and Endocrinology, Japan.

Objective and Methods An SGLT2 inhibitor (ipragliflozin, dapagliflozin, luseogliflozin, tofogliflozin, or canagliflozin) was administered to 132 outpatients with type 2 diabetes mellitus with or without other antidiabetic drugs for 6 months to evaluate its efficacy, the incidence of adverse events, and its influence on the renal function. Results The patient's mean glycated hemoglobin level significantly improved from 7.52±1.16% to 6.95±0.98% (p<0.001). The body weight of the patients was significantly reduced from 78.0±15.3 kg to 75.6±15.1 kg (p<0.001). The estimated visceral fat area was also significantly reduced from 108.4±44.6 cm to 94.5±45.3 cm (p<0.001). The waist circumference, blood pressure, serum alanine aminotransferase, γ-glutamyl transpeptidase, and uric acid levels also showed a significant decrease. The urinary albumin/creatinine ratio (U-ACR) was significantly reduced in the patients whose U-ACR levels were 30-300 mg/gCr at the baseline. The mean eGFR significantly decreased in the patients with a pre-treatment eGFR value of ≥90 mL/min/1.73 m but remained unchanged in the patients with a pre-treatment value of <90 mL/min/1.73 m. A total of 13 adverse events were noted, including systemic eruption (n=1), cystitis (n=2), pudendal pruritus (n=2), nausea (n=1), malaise (n=1), a strong hunger sensation and increased food ingestion (n=1), and non-serious hypoglycemia (n=5). Conclusion SGLT2 inhibitors seemed to be useful in the treatment of obese type 2 diabetes mellitus patients. Furthermore, these data suggest that SGLT2 inhibitors may protect the renal function.
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http://dx.doi.org/10.2169/internalmedicine.56.7196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410466PMC
May 2017

In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models.

J Virol 2016 09 12;90(17):8005-12. Epub 2016 Aug 12.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Unlabelled: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo

Importance: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.
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http://dx.doi.org/10.1128/JVI.00973-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988146PMC
September 2016

Induction of ovine trophoblast cell fusion by fematrin-1 in vitro.

Anim Sci J 2016 Mar 24;87(3):419-22. Epub 2015 Jul 24.

Laboratory of Veterinary Physiology, Co-Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka, Japan.

Endogenous retroviruses present in the genomes take a specific role in placental formation in various vertebrates, including bovine and sheep. Fematrin-1, which is the envelope (Env) protein of bovine endogenous retrovirus found in bovine placenta, is involved in the formation of fetomaternal hybrid cells in cattle placenta. This study was conducted to clarify whether fematrin-1 possesses fusogenic activity in trophoblast cells. Another question is whether Env proteins only have species-specific activity or not. For this, fematrin-1 gene was transfected in ovine trophoblast cells, and we examined fusogenic activity with Cos-7 cells. Although fematrin-1 fusogenic activity was detected in both neutral and acidic pH conditions, acidic condition significantly enhanced it. These activities were rather weaker than those of vesicular stomatitis virus G protein as a positive control. However, the ratio of fematrin-1 and vesicular stomatitis virus G protein fusion index was confirmed similar to those in the previous reports. Some fusion cells showed multinucleate cells. These results imply that fematrin-1 is involved in the formation of trophoblast hybrid cells even in different species trophoblastic cells.
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http://dx.doi.org/10.1111/asj.12439DOI Listing
March 2016

The Roles of Syncytin-Like Proteins in Ruminant Placentation.

Viruses 2015 Jun 5;7(6):2928-42. Epub 2015 Jun 5.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan.

Recent developments in genome sequencing techniques have led to the identification of huge numbers of endogenous retroviruses (ERV) in various mammals. ERVs, which occupy 8%-13% of mammalian genomes, are believed to affect mammalian evolution and biological diversity. Although the functional significance of most ERVs remains to be elucidated, several ERVs are thought to have pivotal roles in host physiology. We and other groups recently identified ERV envelope proteins (e.g., Fematrin-1, Syncytin-Rum1, endogenous Jaagsiekte sheep retrovirus Env) that may determine the morphogenesis of the unique fused trophoblast cells, termed trinucleate cells and syncytial plaques, found in ruminant placentas; however, there are still a number of outstanding issues with regard to the role of ERVs that remain to be resolved. Here, we review what is known about how these ERVs have contributed to the development of ruminant-specific trophoblast cells.
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http://dx.doi.org/10.3390/v7062753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488720PMC
June 2015

Susceptibility of domestic animals to a pseudotype virus bearing RD-114 virus envelope protein.

Gene 2015 Aug 30;567(2):189-95. Epub 2015 Apr 30.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan; Laboratory of Virolution, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address:

Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog.
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http://dx.doi.org/10.1016/j.gene.2015.04.079DOI Listing
August 2015

Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

J Virol 2014 Jun 2;88(12):6896-905. Epub 2014 Apr 2.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan

Unlabelled: Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs.

Importance: Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry receptor and virus-host membrane fusion, respectively. However, unexplained issues remain for the maturation process of retroviral Env. We previously reported that bovine endogenous retrovirus K2 (BERV-K2) Env lost fusogenicity due to a defect in the cleavage of SU and TM. In this study, we identified that mutations residing in BERV-K2 SU disturbed intracellular trafficking of BERV-K2 Env and resulted its inefficient cleavage. Because SU is not known to play an important role in this process, our study may provide novel insights into the maturation mechanism of retroviral Envs.
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http://dx.doi.org/10.1128/JVI.00288-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054351PMC
June 2014

Fematrin-1 is involved in fetomaternal cell-to-cell fusion in Bovinae placenta and has contributed to diversity of ruminant placentation.

J Virol 2013 Oct 17;87(19):10563-72. Epub 2013 Jul 17.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan.

During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin-1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution.
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http://dx.doi.org/10.1128/JVI.01398-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807419PMC
October 2013

Stereocontrolled synthesis of trichodermatide A.

Angew Chem Int Ed Engl 2013 Mar 18;52(13):3646-9. Epub 2013 Feb 18.

Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.

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http://dx.doi.org/10.1002/anie.201210099DOI Listing
March 2013

Dynamic evolution of endogenous retrovirus-derived genes expressed in bovine conceptuses during the period of placentation.

Genome Biol Evol 2013 ;5(2):296-306

Center for Information Biology, National Institute of Genetics, Japan.

In evolution of mammals, some of essential genes for placental development are known to be of retroviral origin, as syncytin-1 derived from an envelope (env) gene of an endogenous retrovirus (ERV) aids in the cell fusion of placenta in humans. Although the placenta serves the same function in all placental mammals, env-derived genes responsible for trophoblast cell fusion and maternal immune tolerance differ among species and remain largely unidentified in the bovine species. To examine env-derived genes playing a role in the bovine placental development comprehensively, we determined the transcriptomic profiles of bovine conceptuses during three crucial windows of implantation periods using a high-throughput sequencer. The sequence reads were mapped into the bovine genome, in which ERV candidates were annotated using RetroTector(©) (7,624 and 1,542 for ERV-derived and env-derived genes, respectively). The mapped reads showed that approximately 18% (284 genes) of env-derived genes in the genome were expressed during placenta formation, and approximately 4% (63 genes) were detected for all days examined. We verified three env-derived genes that are expressed in trophoblast cells by polymerase chain reaction. Out of these three, the sequence of env-derived gene with the longest open reading frame (named BERV-P env) was found to show high expression levels in trophoblast cell lines and to be similar to those of syncytin-Car1 genes found in dogs and cats, despite their disparate origins. These results suggest that placentation depends on various retrovirus-derived genes that could have replaced endogenous predecessors during evolution.
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http://dx.doi.org/10.1093/gbe/evt007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590765PMC
September 2013

Identification and functional analysis of three isoforms of bovine BST-2.

PLoS One 2012 20;7(7):e41483. Epub 2012 Jul 20.

Department of Emerging Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041483PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401110PMC
February 2013

Binding of transcription factor activating protein 2 γ on the 5'-proximal promoter region of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B.

Xenotransplantation 2012 May-Jun;19(3):177-85

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan.

Background: Xenotransplantation is one of the solutions for the shortage of organ donors, and pigs have been considered to be the most suitable animal donors. Specific pathogen-free pigs are utilized in the xenotransplantation; however, pigs have infectious gammaretroviruses, named porcine endogenous retroviruses (PERVs) in their genome. Of them, PERV-A and PERV-B can infect human cells in vitro and potentially induce diseases like other gammaretroviruses. The human cellular receptors for PERV-A were identified and named human PERV-A receptor (HuPAR)-1 and HuPAR-2 (also called as GPR172A and GPR172B, respectively). We have recently reported that HuPAR-2 expression was regulated by epigenetic modification and preferentially expressed in placenta. However, the detailed mechanisms of HuPAR-2 expression have not been fully characterized. In this study, we analyzed molecular mechanisms associated with HuPAR-2 transcription through the identification of transcription factors that bind to the promoter region of HuPAR-2.

Methods: In situ hybridization was performed to identify the cells expressing HuPAR-2 in placental tissues. Transcriptional activities were measured by dual-luciferase reporter assay using serial deletion mutants of HuPAR-2 5'-flanking region. To identify the transcription factors bound to the promoter region, in silico analysis, electrophoresis mobility shift assay, and chromatin immunoprecipitation assay were conducted. The effect of the transcription factor transcription factor activator protein (TFAP)-2γ on the promoter activities was investigated by overexpression of the factor.

Results: We identified that HuPAR-2 was specifically expressed in villous trophoblast cells. We also identified that a region spanning from -126 to -32 had proximal promoter activities and TFAP-2γ bound to a region spanning from -58 to -35 in vitro and in vivo. The overexpression of TFAP-2γ also augmented the proximal promoter activity.

Conclusion: We demonstrated that TFAP-2γ is one of the transcription factors involved in the HuPAR-2 expression in human villous trophoblast cells. By studying transcriptional factors involved in the expression of HuPAR-2, we may find a clue to control the potential risks caused by PERV-A infection in xenotransplantation.
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http://dx.doi.org/10.1111/j.1399-3089.2012.00701.xDOI Listing
October 2012

Sequence comparison of three infectious molecular clones of RD-114 virus.

Virus Genes 2012 Oct 26;45(2):393-7. Epub 2012 May 26.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan.

RD-114 virus is a replication-competent feline endogenous retrovirus. RD-114 virus contaminates several feline and canine live attenuated vaccines and the issue of contamination of RD-114 virus in vaccines should be solved. To date, three infectious molecular clones (pSc3c, pCRT1, and pRD-UCL) have been reported. In this study, we sequenced the entire nucleotide sequence of pRD-UCL and compared the nucleotide sequences of the three infectious molecular clones. As a result, these three infectious clones were nearly identical with each other in gag-pol and env coding regions. These data support the notion that the active locus of infectious RD-114 virus is single in the feline genome. The length of long terminal repeat (LTR) of pCRT1 was 47 bp shorter than those of pSc3c and pRD-UCL. The 47-bp sequence named direct repeat A (DR-A) was duplicated in the U3 region in pSc3c and pRD-UCL. Although several potential enhancer binding sites are present in the DR-A, there was no significant difference in promoter activities between the LTRs of pRD-UCL and pCRT1 in two human cell lines. We also analyzed the splicing pattern of the RD-114 virus by reverse transcription-polymerase chain reaction and confirmed that RD-114 virus is a simple retrovirus. The data presented here will provide basic information about RD-114 virus to solve the contamination issue in live attenuated vaccines.
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http://dx.doi.org/10.1007/s11262-012-0759-0DOI Listing
October 2012

Bovine trophoblastic cell differentiation and binucleation involves enhanced endogenous retrovirus element expression.

Reprod Biol Endocrinol 2012 May 25;10:41. Epub 2012 May 25.

Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.

Background: Endogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines.

Methods: In this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE-B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry.

Results: bERVE-A, bERVE-B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P < 0.01).

Conclusions: These results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.
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http://dx.doi.org/10.1186/1477-7827-10-41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419082PMC
May 2012

Mapping of a neutralizing epitope in the surface envelope protein of porcine endogenous retrovirus subgroup B.

J Gen Virol 2011 Apr 12;92(Pt 4):940-4. Epub 2011 Jan 12.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan.

Pigs are thought to be the most suitable donor animal for xenotransplantation. However, pigs harbour potentially hazardous infectious agents, termed porcine endogenous retroviruses (PERVs), in their genome. In this study, we generated a mAb against PERV-B surface (SU) envelope protein (Env), designated KRT1. KRT1 binding was detected by an indirect immunofluorescence assay and flow cytometric analysis on cells infected with PERV-B. KRT1 neutralized PERV-B pseudotype virus and specifically recognized PERV-B SU Env, but not PERV-A SU Env by immunoblotting analysis. The peptide-ELISA revealed that KRT1 recognized a linear peptide sequence (ALEPPHNLPVP) residing in a proline-rich region that is one of the subdomains of SU Env. In conclusion, the KRT1 antibody will serve as a useful tool for the study of PERV-B and, more importantly, it may provide new protective strategies against PERV-B infection in xenotransplantation.
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http://dx.doi.org/10.1099/vir.0.029322-0DOI Listing
April 2011

Identification of novel endogenous betaretroviruses which are transcribed in the bovine placenta.

J Virol 2011 Feb 17;85(3):1237-45. Epub 2010 Nov 17.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto-city, Kyoto 606-8507, Japan.

Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3' long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.
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http://dx.doi.org/10.1128/JVI.01234-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020495PMC
February 2011

Epigenetic regulation on the 5'-proximal CpG island of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B.

Microbes Infect 2011 Jan 14;13(1):49-57. Epub 2010 Oct 14.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto, Japan.

Porcine endogenous retroviruses (PERVs) have been considered one of the major risks of xenotransplantation from pigs to humans. PERV-A efficiently utilizes human PERV-A receptor 2 (HuPAR-2)/GPR172B to infect human cells; however, there has been no study on the regulation mechanisms of HuPAR-2/GPR172B expression. In this study, we examined the expression of HuPAR-2/GPR172B from the standpoint of epigenetic regulation and discussed the risks of PERV-A infection in xenotransplantation. Quantitative real-time RT-PCR revealed that HuPAR-2 mRNA was preferentially expressed in placental tissue, whereas it was highly suppressed in BeWo cells (a human choriocarcinoma cell line) and HEK293 cells. A CpG island containing the HuPAR-2 transcription starting site was identified by in silico analysis. The DNA methylation ratio (the relative quantity of methylcytosine to total cytosine) and histone modification (H3K9me3) levels in the CpG island measured by bisulfite genomic sequencing and ChIP assay, respectively, were inversely correlated with the mRNA levels. Both HuPAR-2 mRNA and HuPAR-2 protein were up-regulated in HEK293 cells by inhibiting DNA methylation and histone deacetylation. Additionally, promoter/enhancer activities within the CpG island were suppressed by in vitro DNA methylation. Our results demonstrated that epigenetic modification regulates HuPAR-2 expression.
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http://dx.doi.org/10.1016/j.micinf.2010.09.014DOI Listing
January 2011

Unusual permeability of porcine endogenous retrovirus subgroup A through membrane filters.

J Vet Med Sci 2010 Jan 13;72(1):67-71. Epub 2009 Nov 13.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan.

In xenotransplantation from pigs to humans, a bio-artificial endocrine pancreas (Bio-AEP), in which pancreatic endocrine cells are encapsulated within a semipermeable membrane of 100 nm pore size, has been developed. We evaluated the permeability of porcine endogenous retroviruses (PERVs) through membrane filters using a pseudotype virus (LacZ(PERV-A)) containing a viral core derived from murine leukemia virus and an envelope (Env) from PERV subgroup A. Contrary to our expectations, LacZ(PERV-A) lost its infectivity by filtration through a 200 nm membrane filter. This unusual phenotype was not observed in pseudotype viruses harboring Envs from other gammaretroviruses. The infectivity of LacZ(PERV-A) was significantly decreased by repeated freeze/thaw treatment, indicating that LacZ(PERV-A) was physically labile. In addition, LacZ(PERV-A) may be agglutinated because copy numbers of viral RNA after filtration were significantly reduced by filtration through the 200 nm membrane. This phenotype is advantageous to develop a safe Bio-AEP blocking PERV infection.
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http://dx.doi.org/10.1292/jvms.09-0184DOI Listing
January 2010

Focus assay on FeLIX-dependent feline leukemia virus.

J Vet Med Sci 2010 Jan 13;72(1):117-21. Epub 2009 Nov 13.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan.

T-lymphotropic feline leukemia virus (FeLV-T) induces immunodeficiency in cats. FeLV-T is fusion-defective and requires a cofactor, termed FeLIX, for infection. FeLIX is a truncated envelope glycoprotein of an endogenous FeLV and mediates infection by binding a phosphate transporter Pit-1. In this study, we established a feline sarcoma-positive leukemia-negative cell line expressing FeLIX, named QN/FeLIX cells. Upon infection, FeLV-T induced prominent foci with syncytia in QN/FeLIX cells and could be titrated by the focus assay. In addition, we established a FeLIX-expressing feline fibroblast cell line, named AH/FeLIX cells. FeLV-T productively infected AH/FeLIX cells and induced severe CPE with syncytia. QN/FeLIX and AH/FeLIX cells will be useful for the study of FeLIX-dependent mutants in FeLV-infected cats.
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http://dx.doi.org/10.1292/jvms.09-0194DOI Listing
January 2010

The characterization of DNA methylation-mediated regulation of bovine placental lactogen and bovine prolactin-related protein-1 genes.

BMC Mol Biol 2009 Mar 5;10:19. Epub 2009 Mar 5.

Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan.

Background: Bovine trophoblast binucleate cells (BNC) express a plethora of molecules including bovine placental lactogen (bPL, gene name is bCSH1) and bovine prolactin-related protein-1 (bPRP1). BCSH1 and bPRP1 are members of the growth hormone (GH)/prolactin (PRL) gene family, which are expressed simultaneously in BNC and are central to placentation and the progression of pregnancy in cattle. However, there is a paucity of information on the transcriptional regulatory mechanisms of both the bCSH1 and bPRP1 genes. Recent studies, however, have demonstrated that the expression of a number of genes is controlled by the methylation status of their promoter region. In the present study, we examined the cell-type-specific epigenetic alterations of the 5'-flanking region of the bCSH1 and bPRP1 genes to gain an insight into their regulatory mechanisms.

Results: Analysis of 5-aza-2'-deoxycytidine treatment demonstrated that bCSH1 expression is moderately induced in fibroblast cultures but enhanced in BT-1 cells. Sodium bisulfite based sequencing revealed that bCSH1 is hypomethylated in the cotyledonary tissue but not in the fetal skin, and this pattern was not altered with the progression of pregnancy. On the other hand, the methylation status of bPRP1 was similar between the cotyledon and fetal skin. The bPRP1 gene was exclusively hypermethylated in a bovine trophoblast cell-derived BT-1 cell-line. While the activity of bCSH1 was similar in both BT-1 and bovine fibroblast cells, that of bPRP1 was specific to BT-1. Treatment with a demethylating agent and luciferase assays provided in vitro evidence of the positive regulation of bCSH1 but not bPRP1.

Conclusion: This is the first report to identify the differential regulatory mechanisms of the bCSH1 and bPRP1 genes and indicates that bCSH1 might potentially be the only transcript that is subject to DNA methyltransferase regulation. The data indicates the possibility of novel kinetics of induction of the synchronously expressed BNC-specific bCSH1 and bPRP1 transcripts, which may aid the understanding of the intricate regulation and specific role(s) of these important molecules in bovine placentogenesis and the progression of pregnancy.
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http://dx.doi.org/10.1186/1471-2199-10-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666728PMC
March 2009