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1.
J Clin Biochem Nutr ; 66(1): 36-42, 2020 Jan.
Article in English | MEDLINE | ID: mdl-32001954

ABSTRACT

This study was conducted to evaluate the regulation mechanism of influenza virus replication following treatment of Madin-Darby canine kidney cells with the soy isoflavone daidzein. We performed comparative qualitative and quantitative analyses of lipid peroxide between mock-infected and virus-infected cells treated with or without daidzein, as it had been reported that daidzein was an antioxidant and lipid peroxide levels increased upon virus infection. Contrary to our belief, lipid peroxides were not elevated in virus-infected cells and no decrease in lipid peroxides was observed in daidzein-treated cells. In daidzein-treated cells, 5-hydroxyeicosatetraenoic acid, the 5-lipoxygenase product derived from arachidonate, was significantly elevated compared to other lipid peroxides. Zileuton (5-lipoxygenase inhibitor) and 5-lipoxygenase knockdown reduced the daidzein-induced antiviral effect. Moreover, virus replication was regulated by treatment with 5-hydroperoxyeicosatetraenoic acid, a precursor of 5-hydroxyeicosatetraenoic acid and 5-lipoxygenase primary product. These results suggest that daidzein regulates virus replication via signal transduction through 5-lipoxygenase products.

2.
Plant Foods Hum Nutr ; 74(4): 538-543, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31728799

ABSTRACT

Our previous study showed anti-influenza virus activity in adlay tea prepared from adlay seeds, naked barley seeds, soybean, and cassia seeds. In this study, we evaluated the anti-influenza virus activity of each component of this tea and analyzed their active ingredients. Each component was roasted and extracted in hot water; the extracts were tested for antiviral activity and their mechanisms of action were studied. All the tea components showed antiviral activity against the H1N1 and H3N2 influenza subtypes and against influenza B. The viral stages inhibited by the components were virus adsorption and replication in proliferative process, suggesting that the action mechanisms of the components might differ from those of oseltamivir acid. Of the tea components, soybean showed the strongest activity. Therefore, we analyzed its active ingredients by liquid chromatography quadruple time-of-flight mass spectrometry (LC/qTOF-MS) and daidzein and glycitein were detected as active ingredients. Here, anti-influenza virus action of glycitein was the first report.


Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human , Humans , Influenza A Virus, H3N2 Subtype , Tea , Virus Replication
3.
J Sci Food Agric ; 98(5): 1899-1905, 2018 Mar.
Article in English | MEDLINE | ID: mdl-28902408

ABSTRACT

BACKGROUND: The present study was conducted aiming to examine the antiviral activity of adlay tea and its components against influenza viruses. We further aimed to clarify the mechanism by which these components regulate virus replication. RESULTS: Adlay tea at a concentration suitable for drinking inhibited the multiplication of influenza viruses. Moreover, our results suggest that individual components of the tea had antiviral activities against the influenza A/PR/8/34 virus. Adlay tea inhibited multiplication of the H1N1, H3N2 and B types of influenza virus, including oseltamivir-resistant viruses. In addition, adlay tea inhibited influenza infection during the periods of virus adsorption to the cell and virus replication. Adlay tea did not suppress hemagglutination inhibition or cell fusion, although it slightly inhibited virus binding to Malin Darby canine kidney cells. Furthermore, our findings suggest that the antiviral compounds included in adlay tea were ingredients other than polyphenols and that there were several types of effective compounds in adlay tea inhibiting several steps of viral replication. CONCLUSION: The results of the present study demonstrate that adlay tea had antiviral effects against influenza viruses. Our findings with respect to adlay tea suggest that the polyphenols might have a small influence on its antiviral activity and that other ingredients might have more influence. © 2017 Society of Chemical Industry.


Subject(s)
Antiviral Agents/pharmacology , Coix/chemistry , Influenza A virus/drug effects , Influenza B virus/drug effects , Influenza, Human/virology , Plant Preparations/pharmacology , Virus Replication/drug effects , Animals , Dogs , Humans , Influenza A virus/genetics , Influenza A virus/physiology , Influenza B virus/genetics , Influenza B virus/physiology , Madin Darby Canine Kidney Cells
4.
Intern Med ; 52(5): 547-9, 2013.
Article in English | MEDLINE | ID: mdl-23448762

ABSTRACT

The coexistence of idiopathic thrombocytopenic purpura (ITP) and active ulcerative colitis (UC) has been reported. We herein report a rare case of UC accompanied by ITP and Helicobacter pylori (H. pylori) infection. A female UC patient was diagnosed with ITP. At that time, the UC was almost in remission and we suspected that the ITP was caused by some factor other than UC. Accordingly, we found H. pylori infection and administered H. pylori eradication therapy. Consequently, the patient's serum platelet count recovered dramatically. Our report demonstrates the importance of conducting examinations for H. pylori infection in ITP patients, even those with UC.


Subject(s)
Colitis, Ulcerative/diagnosis , Helicobacter Infections/diagnosis , Helicobacter pylori , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Adult , Colitis, Ulcerative/complications , Female , Helicobacter Infections/complications , Helicobacter pylori/isolation & purification , Humans , Purpura, Thrombocytopenic, Idiopathic/complications
5.
Biopolymers ; 100(4): 366-79, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23494631

ABSTRACT

Ribosome recycling factor (RRF) is essential for bacterial growth. Structural studies revealed that RRF consists of two domains connected by two short polypeptides at the hinge region. Here, we evaluated the intrinsic stabilities (ΔG*s) of the two domains and the free energy of the domain-domain interactions (ΔG(int)) for mesophilic RRF (RRF from Escherichia coli, EcRRF) and thermophilic RRF (RRF from Thermus thermophilus, TtRRF) by using differential scanning calorimetry and circular dichroic spectroscopy. Despite single endothermic peaks, a higher than unity value for the ratio of calorimetric enthalpy to van't Hoff enthalpy of the unfolding indicated the presence of unfolding intermediates for both RRFs. Deconvolution analysis based on statistical thermodynamics employing multiple states of the unfolding process with domain-domain interactions allowed us to determine ΔG*s of each domain and ΔG(int). The results indicated that domain I has a higher unfolding temperature than domain II and that it stabilizes domain II through ΔG(int), giving rise to an apparent single peak of unfolding. Interestingly, the estimated ΔG(int) values of 6.28 kJ/mol for EcRRF and 26.28 kJ/mol for TtRRF reflect the observation that only EcRRF has recycling activity at ambient temperature. Our present study suggests the importance of a moderate ΔG(int) for biological activity of multidomain proteins.


Subject(s)
Thermodynamics , Thermus thermophilus , Calorimetry , Calorimetry, Differential Scanning , Escherichia coli , Protein Denaturation
6.
Mol Cell Probes ; 24(4): 167-77, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20083192

ABSTRACT

A single-nucleotide polymorphism detection assay using PCR with quenching probes (QP-PCR) was developed for the rapid detection of antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). The mutations examined were in the HHV-6 U69 gene, and were single-base mutations in sequences known to be associated with ganciclovir (GCV) resistance in HCMV. We previously confirmed that they conferred GCV resistance to recombinant baculoviruses (Nakano et al., J. Virol. Methods 161:223-230, 2009). Six characterized mutations, including a previously reported one that encodes a GCV-sensitive kinase-activity mutant (Isegawa et al., J. Clin. Virol. 44:15-19, 2009), were used. The six mutations were separated into three groups based on their location in the U69 protein, and detected by the hybridization of three probes. We developed and validated a set of assays for these mutations using PCR followed by differential melting of a fluorescently labeled oligo probe, on a Roche Light Cycler platform. Nucleobase quenching was used to detect the hybridized probe. The optimized assay could distinguish the different mutants, and easily detected mutants representing 30% of the DNA in a mixed sample. This QP-PCR assay permitted the rapid (1.5 h), objective, and reproducible detection of drug-resistant mutations of HHV-6.


Subject(s)
DNA Probes/metabolism , Drug Resistance, Viral/genetics , Ganciclovir/pharmacology , Genes, Viral/genetics , Herpesvirus 6, Human/genetics , Mutation/genetics , Polymerase Chain Reaction/methods , Antiviral Agents/pharmacology , Child, Preschool , Drug Resistance, Viral/drug effects , Humans , Male , Mutant Proteins/chemistry , Mutant Proteins/genetics , Nucleic Acid Denaturation/drug effects , Nucleotides/genetics , Sensitivity and Specificity , Transition Temperature/drug effects , Viral Proteins/chemistry , Viral Proteins/genetics
7.
J Virol Methods ; 161(2): 223-30, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19559728

ABSTRACT

A denaturing high-performance liquid chromatography (dHPLC) assay was developed to detect antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). Recombinant baculoviruses were created that contained wild-type and mutant forms of the HHV-6 U69 gene, which determines sensitivity to the antiviral drug ganciclovir (GCV). The mutations causing GCV resistance in HHV-6 U69 were single-base mutations adapted from known GCV-resistant DNA sequences of HCMV, and their ability to confer GCV resistance on recombinant baculoviruses was confirmed. Six characterized mutant sequences, including one reported previously that encodes a GCV-sensitive kinase-activity mutant, were used. DNA was extracted, and the levels of homoduplex and heteroduplex DNA in the PCR products from mixed wild-type and mutant viral DNAs were determined using dHPLC. The optimized assay could distinguish the different mutants, and could detect mutants representing only 10% of the DNAs. The new assay with dHPLC readout permitted the rapid (4 h), objective, and reproducible detection of HHV-6 drug-resistance mutations.


Subject(s)
Antiviral Agents/therapeutic use , Drug Resistance, Viral/genetics , Ganciclovir/therapeutic use , Genes, Viral , Herpesvirus 6, Human/genetics , Point Mutation , Roseolovirus Infections/virology , Base Sequence , Chromatography, High Pressure Liquid/methods , DNA, Viral/chemistry , DNA, Viral/genetics , Herpesvirus 6, Human/chemistry , Herpesvirus 6, Human/drug effects , Humans , Molecular Sequence Data , Reproducibility of Results , Roseolovirus Infections/drug therapy , Sensitivity and Specificity
8.
J Clin Virol ; 44(1): 15-9, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18952495

ABSTRACT

BACKGROUND: Viral resistance to antiviral drugs can cause serious complications in immunosuppressed patients. We isolated from an allogeneic stem cell transplant (SCT) recipient an antiviral-resistant human herpesvirus 6 (HHV-6) strain with mutations that caused amino acid substitutions. OBJECTIVE: To study the impact of mutations in the U38 and U69 genes of the ganciclovir (GCV)-resistant HHV-6 strain associated with the death of the SCT recipient. STUDY DESIGN: Viruses were obtained from blood taken during symptomatic disease. Mutations in the genes for U69 protein kinase and U38 DNA polymerase were analyzed and the effects of the U69 mutations on GCV resistance were assayed using a recombinant baculovirus system. RESULTS: Increasing HHV-6 antigenemia occurred after 2-3 months of preemptive GCV therapy, followed by symptomatic HHV-6 disease that ended in fatal fungus-related septic shock. The HHV-6 strain isolated from the patient was 100-fold more resistant to GCV than was a wild-type strain. New mutations were found in HHV-6 genes U38 (P462S and A565V) and U69 (L202I and L213I). The mutation of U38 P462S corresponds to a mutation in the UL54 gene (P522S) of a GCV-resistant HCMV. The U69 mutations did not alter GCV sensitivity in baculovirus GCV-resistant assay system. CONCLUSIONS: Drug-resistant mutations arising during preemptive therapy may complicate post-transplant HHV-6 disease in SCT recipients. The increased copy number during GCV treatment of this new GCV-resistant HHV-6 strain correlated with mutations in the U69 and U38 genes. Since the kinase mutation did not alter sensitivity to GCV when tested in the in vitro system, it is likely that the substitutions in the polymerase related to GCV resistance.


Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Ganciclovir/pharmacology , Herpesvirus 6, Human/drug effects , Herpesvirus 6, Human/isolation & purification , Roseolovirus Infections/virology , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Baculoviridae/genetics , Cell Line , Child, Preschool , DNA, Viral/chemistry , DNA, Viral/genetics , Fatal Outcome , Herpesvirus 6, Human/genetics , Humans , Male , Molecular Sequence Data , Mutation, Missense , Sequence Alignment , Sequence Analysis, DNA , Stem Cell Transplantation/adverse effects , Transplantation , Viral Nonstructural Proteins/genetics
9.
J Virol ; 82(2): 710-8, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18003734

ABSTRACT

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.


Subject(s)
Herpesvirus 6, Human/physiology , Nuclear Localization Signals , Phosphotransferases (Alcohol Group Acceptor)/analysis , Phosphotransferases (Alcohol Group Acceptor)/physiology , Active Transport, Cell Nucleus/physiology , Cell Line , Cell Nucleus/chemistry , Cells, Cultured , Glutathione Transferase/analysis , Glutathione Transferase/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Herpesvirus 6, Human/genetics , Humans , Microscopy, Fluorescence , Phosphotransferases (Alcohol Group Acceptor)/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Sequence Deletion
10.
J Virol Methods ; 140(1-2): 25-31, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17156861

ABSTRACT

A quantitative real-time PCR assay was developed to determine the antiviral drug susceptibility of human herpesvirus 6 (HHV-6). After short-term culture of the virus, HHV-6 isolates' susceptibility to the antiviral ganciclovir (GCV) was determined by measuring the HHV-6 variant B (HHV-6B) DNA levels in culture supernatants and infected cells using real-time PCR. A total of 12 well-characterized GCV-sensitive or -resistant strains and clinical isolates were used. This new assay with real-time PCR readout permitted the rapid (3 days), objective, and reproducible determination of HHV-6 drug susceptibilities with no need for stringent control of the initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (rs=0.95) with those from the conventional TCID50 (50% tissue culture infecting dose) reduction assay (TRA). Thus, the real-time PCR assay described in this report was found to be a suitable quantitative method for determining the susceptibility of HHV-6 to antiviral drugs. It is faster and simpler than the TRA, and it is amenable to use in the routine diagnostic virology laboratory.


Subject(s)
Antiviral Agents/pharmacology , Ganciclovir/pharmacology , Herpesvirus 6, Human/drug effects , Polymerase Chain Reaction/methods , Cell Line, Tumor , DNA, Viral/analysis , Herpesvirus 6, Human/classification , Herpesvirus 6, Human/isolation & purification , Humans , Reproducibility of Results , Time Factors
11.
J Biol Chem ; 280(7): 5527-32, 2005 Feb 18.
Article in English | MEDLINE | ID: mdl-15598654

ABSTRACT

Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.


Subject(s)
Amino Acids/metabolism , Bacteria/enzymology , Cytochrome c Group/chemistry , Cytochrome c Group/metabolism , Amino Acids/genetics , Bacteria/genetics , Circular Dichroism , Cytochrome c Group/genetics , Electrochemistry , Enzyme Stability , Escherichia coli/cytology , Escherichia coli/metabolism , Guanidine/pharmacology , Magnetic Resonance Spectroscopy , Mutation/genetics , Periplasm/metabolism , Protein Conformation/drug effects , Protein Denaturation/drug effects , Temperature , Thermodynamics
12.
J Am Chem Soc ; 126(45): 14684-5, 2004 Nov 17.
Article in English | MEDLINE | ID: mdl-15535669

ABSTRACT

The complete thermal-unfolding profiles of both oxidized and reduced forms of cytochrome c551 (PA) from mesophilic Pseudomonas aeruginosa and cytochrome c552 (HT) from thermophilic Hydrogenobacter thermophilus were obtained by the newly developed pressure-proof cell compartment installed in a circular dichroic spectrometer, which facilitates protein thermal-unfolding experiments up to 180 degrees C. The thermodynamic cycle, which relates protein stability and redox function, indicated that the redox potentials of PA and HT in the native state are regulated by the stability of the oxidized proteins rather than by that of the reduced ones.


Subject(s)
Cytochromes c/chemistry , Circular Dichroism , Cytochromes c/metabolism , Hot Temperature , Oxidation-Reduction , Protein Folding , Thermodynamics
13.
J Biol Chem ; 278(5): 3427-36, 2003 Jan 31.
Article in English | MEDLINE | ID: mdl-12411440

ABSTRACT

X-ray and NMR analyses on ribosome recycling factors (RRFs) from thermophilic bacteria showed that they display a tRNA-like L-shaped conformation consisting of two domains. Since then, it has been accepted that domain I, consisting of a three-helix bundle, corresponds to the anticodon arm of tRNA and domain II and a beta/alpha/beta sandwich structure, corresponds to the acceptor arm. In this study, we obtained a RRF from a mesophilic bacterium, Vibrio parahaemolyticus, by gene cloning and carried out an x-ray analysis on it at 2.2 A resolution. This RRF was shown to be active in an in vitro assay system using Escherichia coli polysomes and elongation factor G (EF-G). In contrast, the above-mentioned RRFs from thermophilic bacteria were inactive in such a system. Analysis of the relative orientations between the two domains in the structures of various RRFs, including this RRF from mesophilic bacterium, revealed that domain II rotates about the long axis of the helix bundle of domain I. To elucidate the ribosome binding site of RRF, the peptide fragment (RRF-DI) corresponding to domain I of RRF was expressed and characterized. RRF-DI is bound to 70 S ribosome and the 50 S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30 S subunit. These findings caused us to reinvestigate the concept of the mimicry of RRF to tRNA and to propose a new model where domain I corresponds to the acceptor arm of tRNA and domain II corresponds to the anticodon arm. This is just the reverse of a model that is now widely accepted. However, the new model is in better agreement with published biological findings.


Subject(s)
Proteins/metabolism , Ribosomes/metabolism , Vibrio parahaemolyticus/metabolism , Amino Acid Sequence , Bacteria/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Kinetics , Models, Molecular , Molecular Sequence Data , Polyribosomes/metabolism , Polyribosomes/ultrastructure , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Ribosomal Proteins , Ribosomes/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid
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