[Show abstract][Hide abstract] ABSTRACT: Antiviral drugs are being used for therapeutic purposes against influenza illness in humans. However, antiviral-resistant variants often nullify the effectiveness of antivirals. Combined medications, as seen in the treatment of cancers and other infectious diseases, have been suggested as an option for the control of antiviral-resistant influenza viruses. Here, we evaluated the therapeutic value of combination therapy against oseltamivir-resistant 2009 pandemic influenza H1N1 virus infection in DBA/2 mice. Mice were treated for five days with favipiravir and peramivir starting 4 hours after lethal challenge. Compared with either monotherapy, combination therapy saved more mice from viral lethality and resulted in increased antiviral efficacy in the lungs of infected mice. Furthermore, the synergism between the two antivirals, which was consistent with the survival outcomes of combination therapy, indicated that favipiravir could serve as a critical agent of combination therapy for the control of oseltamivir-resistant strains. Our results provide new insight into the feasibility of favipiravir in combination therapy against oseltamivir-resistant influenza virus infection.
[Show abstract][Hide abstract] ABSTRACT: Axin is a multifunctional protein that participates in many cellular events including Wnt signaling and cell fate determination. Aurora kinase inhibitor (AKI)-induced cell death and cell membrane rupture is facilitated in L929 cells expressing axin (L-axin cells) through the activation of poly ADP-ribose polymerase (PARP). We observed that caspase-2 activity is required for AKI-induced cell death. Inhibition of caspase-2 activity suppressed AKI-induced PARP activation and mitochondrial dysfunction, resulting in a decrease in AKI-induced cell death. When an axin mutant deleted for the glycogen synthase kinase 3β (GSK3β)-binding domain was expressed in L929 cells (L-ΔGSK cells), AKI-induced caspase-2 activation and cell death decreased. AKI treatment reduced the expression of a 32-kDa caspase-2 splicing variant (caspase-2S) in most L-axin cells, but not in L-ΔGSK cells. These results suggest that AKI-induced caspase-2 activation in L-axin cells might be due to a decrease in the expression of caspase-2S, which inhibits caspase-2 activity. In addition, AKI treatment failed to activate caspase-8 and treatment with necrostatin inhibited AKI-induced cell death in L-axin cells, suggesting that the absence of caspase-8 activation might favor necrotic cell death. Axin expression may facilitate AKI-induced caspase-2 activation followed by activation of PARP and initiation of the necrotic cell death pathway.
[Show abstract][Hide abstract] ABSTRACT: Laboratory diagnosis of hemorrhagic fever with renal syndrome (HFRS), an infectious disease caused by rodent-borne hantaviruses in Asia and Europe, depends primarily on serological methods. Since the advent of such serodiagnostic tests, few reports are available about the clinical and molecular epidemiological features of HFRS.
To investigate the epidemioclinical features of HFRS patients treated at a tertiary-care teaching hospital in Seoul over a 10-year period.
Medical records of HFRS patients, admitted to a tertiary-care teaching hospital during February 2002 to February 2012, were reviewed. Sera from patients were tested for Hantaan virus (HTNV) and Seoul virus (SEOV) RNA using RT-PCR.
Among 35 HFRS patients (mean age was 44.2±14.7 years), 29 were male (82.9%). Acute renal failure developed in 27 patients (77.1%), and 12 patients (34.3%) were admitted to the intensive care unit (ICU). Conjunctival injection (OR 10.32, 95% CI 1.09-97.77, P=.04) and initial serum albumin less than 3g/dL (OR 22.83, 95% CI 1.45-359.93, P=.03) were risk factors for ICU admission. Of 35 acute-phase sera, 11 (31.4%) were positive for HTNV RNA. None were positive for SEOV RNA.
HFRS was characterized by the clinical triad of fever, renal insufficiency and gastrointestinal symptoms. Conjunctival injection and serum albumin level were related to severity. A large-scale multi-center study is needed to enhance insights into epidemioclinical characteristics of HFRS in Korea.
Full-text · Article · Jul 2013 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
[Show abstract][Hide abstract] ABSTRACT: Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signaling and participates in various cellular events including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of G-protein signaling (RGS) domain was involved in axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that enhancing viral replication might be mediated by the axin RGS domain.
Full-text · Article · Mar 2013 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.
No preview · Article · Jun 2012 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1-expressing cells (L-Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2-expressing cells (L-Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub-G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L-Axin cells induced poly(ADP-ribose) polymerase (PARP) activation, which increased the poly(ADP-ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI-induced cell death. Also, AKI treatment of L-Axin cells induced mitochondrial apoptosis-inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase-3 activation. Knockdown of AIF attenuated AKI-induced cell death in L-Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition-induced cell death in a PARP- and AIF-dependent manner.
No preview · Article · Sep 2011 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-β and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.
[Show abstract][Hide abstract] ABSTRACT: We have previously identified a defensin-like peptide (BmDp, Genbank accession number DQ118523), in the larvae of a silkworm,
Bombyx mori (B. mori) using wdS20658 cDNA in a Bombyx EST database (Silk Base: http://www.ab.a.u-tokyo.ac.jp/silkbase). A full-length of BmDp cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli cells in order to obtain a functional peptide. Amino acid sequence comparison of cysteine-rich defensin-like peptides with
those of the BmDp showed to be identical protein as BmDefensinA designated as BmdefA gene deposited in the DDBJ database under the accession number AB367525. Sequence alignments revealed that the BmDp belongs
to the insect defensin family and is close to galiomicin and spodoptericin, a defensin and a defensin-like protein having
antimicrobial activities in phylogenetic analysis. Thus, we evaluated the effect of immune challenge and antimicrobial activity
to understand the nature of this anionic BmDp. Semiquantitative RT-PCR analysis showed that BmDp gene expression was inducible
by bacterial injection and peaked at a highest level at 8h after bacterial injection. This result suggests that BmDp is related
with immune response against bacteria. A purified recombinant GSTBmDp fusion protein did not show direct antimicrobial activity
against bacteria and fungi, which is differed from cationic antibacterial insect defensins and neutral antifungal defensin-like
-Defensin-like protein-Antimicrobial activity
[Show abstract][Hide abstract] ABSTRACT: Carbapenem resistance in Acinetobacter baumannii is increasing these days. We investigated the roles of outer membrane proteins and efflux pumps in carbapenem resistance of A. baumannii which showed no carbapenemase activity in modified Hodge test. Among 58 carbapenem-resistant isolates collected from the Korea University Medical Center between January 2002 and March 2006, 17 isolates showed negative results in modified Hodge test. In outer membrane protein analysis, loss of the 29-kDa protein band was related with higher imipenem minimum inhibitory concentrations especially in the presence of OXA-51-like enzymes. Efflux pump-mediated carbapenem resistance was found in one out of the 17 isolates (5.9%). All of the 58 carbapenem-resistant strains and 5 of the 10 carbapenem-susceptible strains had OXA-51-like carbapenemase genes, suggesting that OXA-51-like enzymes may be naturally existing in A. baumannii and have very weak carbapenem hydrolyzing activity.
No preview · Article · Oct 2009 · Microbial drug resistance (Larchmont, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: TheBombyx mori (B. mori) serine protease-like protein (BmSp) coding region (946 bp, GenBank accession number of mRNA, DQ118520; protein, AAZ40503)
was generated from two separate and overlapping cDNA fragments using sequence homology withTrichoplusia ni azurocidin in aBombyx EST database (Silkbase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). The deduced amino acid sequence of BmSp, which encodes
303 amino acids, shows 44% amino acid identity toA. gambiae serine protease (CAA89967), 43% amino acid identity toSarcophagi peregrina 26-kDa protease, an antibacterial protein and 31% identity toB. mori serine protease-2 (BmSP-2), a potential antiviral protein. Typical features of the BmSp included the serine protease active
site triad His / Asp / Ser, three pairs of cysteine residues for disulfide bridges, and three residues, Asp / Gly / Gly, that
help to confer trypsin-like specificity to the enzymes. Based on the result of sequence comparison and characterization, our
results suggest that the BmSp probably the new subfamily of trypsin-like serine protease. Using RT-PCR and enzyme digestion,
the full encoding sequence for BmSp was cloned into theE. coli expression vector pGEX-5X-1. The fusion protein GST-BmSp was effectively expressed inE. coli BL21(DE3) pLysS as inclusion bodies, and a denaturation and refolding procedure were performed to obtain soluble GST-BmSp.
The purified protein was tested for antibacterial activity against Gram-positive and Gram-negative bacteria, but it did not
show antibacterial activity in the agar well diffusion assay and liquid growth inhibition assay.
Key wordsserine protease-
[Show abstract][Hide abstract] ABSTRACT: Soldiers from the Republic of Korea and the United States conducting peacetime military operations at various training sites and multiple range complexes located near the demilitarized zone separating North and South Korea are exposed to rodents and their potentially disease-carrying ectoparasites. These diseases include scrub typhus, murine typhus, and leptospirosis. Many of the training sites are rural or semi-rural, surrounded or co-located with various forms of agriculture, and are infested with rodents and insectivores (as well as their ectoparasites), which are commonly found in association with unmanaged tall grasses, scrub, and crawling vegetation habitats. For 5 years, rodents and insectivores were collected seasonally (spring, summer, fall, and winter) at firing points 10 and 60 near the demilitarized zone and serologically tested for the presence of scrub typhus, murine typhus, and leptospirosis antibodies. Of the nine species of small mammals collected, Apodemus agrarius, the common striped field mouse and known reservoir of scrub typhus, was the most frequently collected (90.6%). Only four of the nine species captured, A. agrarius (60.9%), Micromys minutus (100%), Mus musculus (55.6%), and Rattus norvegicus (46.7%), were positive for scrub typhus. Of all the small mammals captured, only A. agrarius was positive for murine typhus (0.3%) and leptospirosis (1.3%). Seasonal and annual prevalence rates based on weight and sex are presented.
Full-text · Article · May 2009 · Vector borne and zoonotic diseases (Larchmont, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.
Full-text · Article · May 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control.
In the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins
Our results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.
[Show abstract][Hide abstract] ABSTRACT: Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.
No preview · Article · Feb 2009 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Throughout Korea, small mammals are hosts to a number of disease-causing agents that pose a health threat to U.S. and Korean military forces while they conduct field-training exercises. A seasonal rodent-borne disease surveillance program was established at two firing points (FP), FP-10, and FP-60, and conducted over five years from 2001 through 2005 in response to hantavirus cases among U.S. soldiers. The ecology of these sites consisted primarily of tall grasses associated with semi-permanent and temporary water sources (drainage ditches and a small stream) and dry-land agriculture farming. Eight species of rodents and one species of insectivore were collected, including Apodemus agrarius, Micromys minutus, Mus musculus, Rattus norvegicus, Tscherskia triton, Microtus fortis, Myodes regulus, and Crocidura lasiura. The striped field mouse, A. agrarius, (primary reservoir for Hantaan virus, the causative agent of Korean hemorrhagic fever), was the most frequently collected, representing 90.6% of the 1,288 small mammals captured at both sites. Reported herein are the ecological parameters, seasonal population densities, and seasonal population characteristics associated with small mammals collected at two military training sites in the Republic of Korea.
Full-text · Article · Jan 2009 · Journal of Vector Ecology
[Show abstract][Hide abstract] ABSTRACT: Acute-phase sera from >5 % of cases of haemorrhagic fever with renal syndrome occurring annually in Korea have been found to exhibit a fourfold or higher antibody titre to Puumala virus (PUUV) than to Hantaan virus (HTNV) by double-sandwich IgM ELISA, suggesting the existence of a PUUV-related hantavirus. Based on the phylogenetic relationships among arvicolid rodents, the royal vole (Myodes regulus) was targeted as a likely reservoir host of hantavirus. Using RT-PCR, a genetically distinct hantavirus, designated Muju virus (MUJV), was detected in lung tissue of royal voles, captured in widely separated geographical regions in Korea during 1996-2007. Pairwise analysis of the full-length S (1857 nt) and M (3634 nt) segments of MUJV indicated approximately 77 % sequence similarity with PUUV. At the amino acid level, MUJV differed from PUUV by 5.5-6.9 % (nucleocapsid) and 10.0-11.6 % (Gn and Gc envelope glycoproteins). Interstrain variation of MUJV sequences from royal voles captured in different regions suggested geographic-specific clustering. Neutralizing antibody titres against PUUV were two- to sixfold higher than to HTNV in sera of MUJV-infected Myodes regulus. Although virus isolation attempts were unsuccessful, the collective data indicate that MUJV is a distinct hantavirus species.
Full-text · Article · Dec 2007 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Sequence analysis of the full-length medium segment and the partial small and large segments of a hantavirus, detected by reverse transcription-PCR in lung tissues of the Chinese mole shrew (Anourosorex squamipes) captured in Cao Bang Province, Vietnam, in December 2006, indicated that it is genetically distinct from rodentborne hantaviruses.
Full-text · Article · Dec 2007 · Emerging infectious diseases
[Show abstract][Hide abstract] ABSTRACT: A seroepidemiological study of selected rodent-borne diseases (hantavirus [Seoul [SEO] virus], scrub typhus [Orientia tsutsugamushi], murine typhus [Rickettsia typhi], and leptospirosis [Leptospira interrogans]), as part of the U.S. military rodent surveillance and control program, was conducted from 2001 through 2005 at Yongsan Garrison, Seoul, Republic of Korea. Rodents were collected to determine the prevalence of rodent-borne diseases at a U.S. military installation in an urban environment. A total of 1,750 rodents representing three species was collected by using baited live traps (Tomahawk), glue boards, and poison baits (dead rodents observed but not assayed). The Norway rat, Rattus norvegicus (99.8%), accounted for nearly all of the rodents captured/observed. Only three roof rats, Rattus rattus (0.2%), and one house mouse, Mus musculus (<0.1%), were collected. R. norvegicus rats were the only rodents that were serologically positive for SEO virus (9.6%), scrub typhus (2.8%), murine typhus (3.8%), and leptospirosis (4.6%). One of six rodents that were positive for SEO virus by immunofluorescent antibody test was positive for SEO virus antigen by reverse transcriptase-polymerase chain reaction. Infection rates for SEO virus, scrub typhus, murine typhus, and leptospirosis varied annually. Rodents were captured from 228 (20.7%) of 1,104 total buildings in Yongsan Garrison. The Yongsan commissary had the highest annual infestation rate (22 rodents per year), followed by Commisky's Club (18 rodents per year). Annual infestation rates were high for food service facilities, which often store perishable food products outdoors for short periods of time, attracting rodent populations; refuse from these facilities provides harborage and food for rodents. The effect of rodent populations outside the boundary of Yongsan Garrison was not determined.
No preview · Article · Jul 2007 · Military medicine
[Show abstract][Hide abstract] ABSTRACT: Dopamine (DA) is an oxidant that may contribute to the degeneration of dopaminergic neurons. The present study demonstrates that DA-induced cytotoxicity in human-derived neurotypic cells, SH-SY5Y, is prevented by resveratrol, one of the major antioxidative constituents found in the skin of grapes. SH-SY5Y cells, a neuroblastoma cell line, treated with DA at 300 and 500 microM for 24 h underwent apoptotic death as determined by characteristic morphological features, including nuclear condensation, and loss of mitochondrial membrane potential (MMP). Flow cytometric analysis using Annexin V showed that DA can induce significant and severe apoptosis. Exposure to resveratrol (5 microM) for 1 h prior to the DA treatment attenuated DA-induced cytotoxicity, and rescued the loss of MMP. To investigate the apoptotic signaling pathways relevant to the restoration of DA-induced apoptosis by resveratrol, we carried out quantitative analysis of Bcl-2, caspase-3, and cleaved poly ADP-ribose polymerase (PARP) by immunoblot analysis. Resveratrol pretreatment led to a decrease in cleavage of PARP, an increase in the Bcl-2 protein, and activation of caspase-3. These results suggest that DA may be a potential oxidant of neuronal cells at biologically relevant concentrations. Resveratrol may protect SH-SY5Y cells against this cytotoxicity, reducing intracellular oxidative stress through canonical signal pathways of apoptosis and may be of biological importance in the prevention of a dopaminergic neurodegenerative disorder such as Parkinson disease.
Full-text · Article · Jul 2007 · Experimental and Molecular Medicine