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author:("Kim, seen Hee")
1.  Draft Genome Sequence of Petroleum Oil-Degrading Marine Bacterium Pseudomonas taeanensis Strain MS-3, Isolated from a Crude Oil-Contaminated Seashore 
Genome Announcements  2014;2(1):e00818-13.
Pseudomonas taeanensis MS-3T, isolated from a crude oil-contaminated seashore in South Korea, is capable of degrading petroleum oils, such as gasoline, diesel, and kerosene. Here, we report the draft genome sequence of this strain, which consists of 5,477,045 bp, with a G+C content of 60.72%.
doi:10.1128/genomeA.00818-13
PMCID: PMC3886939  PMID: 24407626
2.  Plasma-derived MHCII+ exosomes from tumor-bearing mice suppress tumor antigen-specific immune responses 
European journal of immunology  2012;42(7):1778-1784.
Tumor-specific immunosuppression is frequently observed in tumor-bearing hosts. Exosomes are nano-sized, endosomal-derived membrane vesicles secreted by most tumor and hematopoietic cells and have been shown to actively participate in immune regulation. We previously demonstrated that antigen-specific immunosuppressive exosomes could be isolated from the blood plasma of antigen-immunized mice. Here we demonstrate that plasma-derived exosomes isolated from mice bearing OVA-expressing tumors were able to suppress OVA-specific immune response in a mouse delayed-type hypersensitivity model. Enrichment of tumor-derived exosomes in the plasma of mice bearing subcutaneous melanoma was not detected using an exosome-tagging approach. Instead, depletion of MHC Class II+ vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC Class II deficient mice resulted in significant abrogation of the suppressive effect. These results demonstrate that circulating host-derived, MHC Class II+ exosomes in tumor-bearing hosts are able to suppress the immune response specific to tumor antigens.
doi:10.1002/eji.201141978
PMCID: PMC3471134  PMID: 22585706
3.  Differential Activation of Diverse Glutathione Transferases of Clonorchis sinensis in Response to the Host Bile and Oxidative Stressors 
Background
Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other.
Methodology/Principal Findings
We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses.
Conclusions/Significance
Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.
Author Summary
Clonorchis sinensis is a trematode parasite that infects the hepatobiliary ducts of the mammals including humans. Approximately 35 million people are infected and 600 million people are at risk of infections. Epidemiological studies have convincingly demonstrated relationships between clonorchiasis and cholangiocarcinoma. C. sinensis is a Group 1 biological carcinogen. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship. We observed global expression pattern of C. sinensis ESP in the presence of host bile/oxidative stresses, which mimics natural host environments. The secretory proteome displayed common and unique features, which might be related to its habitat in the definitive host. The universal proteins were found to be several enzymes involved in glucose metabolism, glutathione transferases (GSTs) and oxygen transporters. C. sinensis differentially regulated the secretion of diverse GSTs in response to bile and oxidative stressors, which suggested that these enzymes are importantly involved in the protection from immune cell-derived oxidizing molecules and detoxification of hydrophobic substances. C. sinensis secreted less cysteine proteases, which play roles in tissue migration and immune evasion, compared to other tissue-invasive or intravascular trematodes. Our data suggest strongly that different GSTs might have differentially evolved with their specialized functions to cope with stressful conditions and ensure parasite's long-standing survival in the hosts.
doi:10.1371/journal.pntd.0002211
PMCID: PMC3656158  PMID: 23696907
4.  The Determination of Importance of Sequences Neighboring the Psi Sequence in Lentiviral Vector Transduction and Packaging Efficiency 
PLoS ONE  2012;7(11):e50148.
A number of lentiviral vector systems have been developed for gene delivery and therapy by eliminating and/or modifying viral genetic elements. However, all lentiviral vector systems derived from HIV-1 must have a viral packaging signal sequence, Psi (Ψ), which is placed downstream of 5′ long terminal repeat in a transgene plasmid to effectively package and deliver transgene mRNA. In this study, we examined feasible regions or sequences around Psi that could be manipulated to further modify the packaging sequence. Surprisingly, we found that the sequences immediately upstream of the Psi are highly refractory to any modification and resulted in transgene vectors with very poor gene transduction efficiency. Analysis around the Psi region revealed that there are a few sites that can be used for manipulation of the Psi sequence without disturbing the virus production as well as the efficiency of transgene RNA packaging and gene transduction. By exploiting this new vector system, we investigated the requirement of each of four individual stem-loops of the Psi sequence by deletion mapping analysis and found that all stem-loops, including the SL4 region, are needed for efficient transgene RNA packaging and gene delivery. These results suggest a possible frame of the lentiviral vector that might be useful for further modifying the region/sequence around the packaging sequence as well as directly on the Psi sequence without destroying transduction efficiency.
doi:10.1371/journal.pone.0050148
PMCID: PMC3503997  PMID: 23185560
5.  Exosomes from IDO+ DC are therapeutic in CIA and DTH disease models 
Arthritis and rheumatism  2009;60(2):380-389.
Objective
We have demonstrated previously that dendritic cells (DC), modified with immunosuppressive cytokines, and exosomes derived from the DC can suppress the onset of murine CIA and reduce the severity of established arthritis. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degrading enzyme important for immune regulation and tolerance maintenance. DC expressing functional IDO can inhibit T cells by either depleting them of essential tryptophan and/or by producing toxic metabolites, as well as by generating regulatory T cells. In this study, we examined the immunosuppressive effects of bone marrow derived DC, genetically modified to express IDO, and IDO+-DC-derived exosomes.
Methods
Bone marrow derived DC were adenovirally transduced with IDO or CTLA4-Ig (an inducer of IDO), and the resulting DC and exosomes were tested for their immunosuppressive ability in the collagen-induced arthritis and delayed type hypersensitivity murine models.
Results
We demonstrate that both DC and exosomes derived from DC overexpressing IDO are anti-inflammatory in collagen-induced arthritis and delayed type hypersensitivity murine models. The suppressive effects were partially dependent on B7 costimulatory molecules. In addition, gene transfer of CTLA4-Ig to DC resulted in induction of IDO in the DC and exosomes able to reduce inflammation in an IDO-dependent manner.
Conclusion
These results demonstrate that both IDO expressing DC and DC-derived exosomes are immunosuppressive and anti-inflammatory, and are able to reverse established arthritis. Therefore, exosomes from IDO+ DC may represent a novel therapy for rheumatoid arthritis.
doi:10.1002/art.24229
PMCID: PMC3491653  PMID: 19180475
Dendritic cells; Exosomes; IDO; Arthritis; Inflammatory disease
6.  Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode 
Background
Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive.
Methodology/Principal Findings
We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol.
Conclusions/Significance
The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.
Author Summary
Neurocysticercosis (NC), an infection of the central nervous system with Taenia solium metacestode (TsM), constitutes a leading cause of adult-onset seizures in endemic areas. Like other helminths, TsM is incapable of synthesizing lipid molecules. It should be equipped with a specialized system for lipid transportation from the host to ensure its long-survival. Such a transport system may be a target for function-associated drug design. We characterized two novel fatty-acid (FA)-binding TsM proteins (TsMFABP1 and TsMFABP2). Native and recombinant proteins bound to several FA analogs and retinol at micromolar and millimolar concentrations. Their binding was specifically inhibited by oleic acid. TsMFABP1exhibited high affinity toward FA analogs, while TsMFABP2 showed preferential affinity to retinol. Both TsMFABPs were predominantly expressed in the canal region of the worm, where lipids and retinol were abundantly distributed. The two paralogous TsMFABPs have undergone (or are still undergoing) structural diversification and following functional divergence to act as FABP or retinol binding protein, similar to the intracellular lipid binding proteins of deuterostomian animals. The canal region specific distribution of lipids, retinol and FABPs further suggested that cells in this area might differentiate into diverse cells to compose huge numbers of the proglottids, thereby playing vital roles in the parasite growth and development.
doi:10.1371/journal.pntd.0001868
PMCID: PMC3493614  PMID: 23150743
7.  Application of Percutaneous Cardiopulmonary Support for Cardiac Tamponade Following Blunt Chest Trauma: Two Case Reports 
Since the advent of percutaneous cardiopulmonary support (PCPS), its application has been extended to massively injured patient. Cardiac injury following blunt chest trauma brings out high mortality and morbidity. In our cases, patients had high injury severity score by blunt trauma and presented sudden hemodynamic collapse in emergency room. We quickly detected cardiac tamponade by focused assessment with sonography for trauma and implemented PCPS. As PCPS established, their vital sign restored and then, they were transferred to the operation room (OR) securely. After all injured lesion repaired, PCPS weaned successfully in OR. They were discharged without complication on day 26 and 55, retrospectively.
doi:10.5090/kjtcs.2012.45.5.334
PMCID: PMC3487020  PMID: 23130310
Extracorporeal circulation; Trauma, blunt; Cardiac tamponade; Ultrasonic diagnosis; Cardiac rupture
8.  Tumor-Derived Exosomes Confer Antigen-Specific Immunosuppression in a Murine Delayed-Type Hypersensitivity Model 
PLoS ONE  2011;6(8):e22517.
Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH) and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA) resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.
doi:10.1371/journal.pone.0022517
PMCID: PMC3149056  PMID: 21829629
9.  B7-1/2, but not PD-L1/2 molecules, are required on IL-10-treated tolerogenic DC and DC-derived exosomes for in vivo function 
European journal of immunology  2009;39(11):3084-3090.
SUMMARY
Costimulatory molecules such as B7-1/2 and PD-L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7-1/2 and PD-L1/2 to the function of IL-10-treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL-10 treatment of DC significantly downregulated surface expression of MHC II, B7-1, B7-2, and decreased levels of MHC I and PD-L2. IL-10 treatment of DC resulted in a modified co-stimulatory profile of DC-secreted exosomes with a reduction in B7-1, PD-L1 and PD-L2. We further demonstrate that absence of B7-1 or B7-2 on donor DC results in a loss of ability of IL-10 treated DC and their exosomes to suppress the delayed-type hypersensitivity (DTH) response, whereas IL-10 treated DC deficient in PD-L1/2 as well as their secreted exosomes retained the ability to suppress DTH responses. We conclude that B7-1 and B7-2, but not PD-L1 and PD-L2, on IL-10 treated DC and DC-derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.
doi:10.1002/eji.200939407
PMCID: PMC3075967  PMID: 19757438
Dendritic cells; Exosomes; B7-1/2; PD-L1/2; Delayed-type hypersensitivity
10.  Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4 
Background
Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.
Findings
We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5–7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.
Conclusion
VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.
Author Summary
Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax.
doi:10.1371/journal.pntd.0000849
PMCID: PMC2953480  PMID: 20967286
11.  Adenoviral Mediated, Intra-Tumor Gene Transfer of Interleukin 23 Induces a Therapeutic Anti-tumor Response 
Cancer gene therapy  2009;16(10):776-785.
IL-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. IL-23 has been shown to possess potent anti-tumor activities in several establishment models of cancer and a few therapeutic models, but the efficacy of local, adenoviral-mediated expression of IL-23 in established tumors has yet to be investigated. Here we have examined the anti-tumor activity of adenovirally-delivered IL-23 in a day 7 MCA205 murine fibrosarcoma tumor model. Three intratumoral injections of adenovirus expressing IL-23 (Ad.IL-23) significantly increased animal survival and resulted in complete rejection of 40 percent of tumors, with subsequent generation of protective immunity and MCA205-specific cytotoxic T-lymphocytes (CTLs). Additionally, we have shown that the anti-tumor activity of IL-23 is independent of IL-17, perforin and Fas ligand, but dependent on IFN-γ, CD4 and CD8 positive T-cells. These results demonstrate that direct intratumoral injection of adenovirus expressing IL-23 results in enhanced survival, tumor eradication and generation of protective immunity by generation of a Th1-type immune response.
doi:10.1038/cgt.2009.27
PMCID: PMC2745505  PMID: 19390568
Interleukin 23; adenovirus; cancer; gene therapy
12.  Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals 
Background
Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown.
Results
We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH)-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx)-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms.
Conclusion
Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic expansion/deletion and exon-intron remodeling. The differentiated enzymatic properties might be acquired by the evolutionary relaxation of selection pressure and/or biochemical adaptation to the acting environments. Our present study would be beneficial to get detailed insights into the complex GPx evolution, and to understand the molecular basis of the specialized physiological implications of this antioxidant system in their respective donor organisms.
doi:10.1186/1471-2148-9-72
PMCID: PMC2679728  PMID: 19344533
13.  PwRn1, a novel Ty3/gypsy-like retrotransposon of Paragonimus westermani: molecular characters and its differentially preserved mobile potential according to host chromosomal polyploidy 
BMC Genomics  2008;9:482.
Background
Retrotransposons have been known to involve in the remodeling and evolution of host genome. These reverse transcribing elements, which show a complex evolutionary pathway with diverse intermediate forms, have been comprehensively analyzed from a wide range of host genomes, while the information remains limited to only a few species in the phylum Platyhelminthes.
Results
A LTR retrotransposon and its homologs with a strong phylogenetic affinity toward CsRn1 of Clonorchis sinensis were isolated from a trematode parasite Paragonimus westermani via a degenerate PCR method and from an insect species Anopheles gambiae by in silico analysis of the whole mosquito genome, respectively. These elements, designated PwRn1 and AgCR-1 – AgCR-14 conserved unique features including a t-RNATrp primer binding site and the unusual CHCC signature of Gag proteins. Their flanking LTRs displayed >97% nucleotide identities and thus, these elements were likely to have expanded recently in the trematode and insect genomes. They evolved heterogeneous expression strategies: a single fused ORF, two separate ORFs with an identical reading frame and two ORFs overlapped by -1 frameshifting. Phylogenetic analyses suggested that the elements with the separate ORFs had evolved from an ancestral form(s) with the overlapped ORFs. The mobile potential of PwRn1 was likely to be maintained differentially in association with the karyotype of host genomes, as was examined by the presence/absence of intergenomic polymorphism and mRNA transcripts.
Conclusion
Our results on the structural diversity of CsRn1-like elements can provide a molecular tool to dissect a more detailed evolutionary episode of LTR retrotransposons. The PwRn1-associated genomic polymorphism, which is substantial in diploids, will also be informative in addressing genomic diversification following inter-/intra-specific hybridization in P. westermani populations.
doi:10.1186/1471-2164-9-482
PMCID: PMC2582038  PMID: 18851759
14.  Characterization of Plasmodium vivax Heat Shock Protein 70 and Evaluation of Its Value for Serodiagnosis of Tertian Malaria† ▿  
Clinical and Vaccine Immunology  2007;14(3):320-322.
We have characterized Plasmodium vivax heat shock protein 70 (PvHSP70) and evaluated serodiagnostic applicability of recombinant PvHSP70 (rPvHSP70). In enzyme-linked immunosorbent assays and immunoblot analyses, rPvHSP70 showed high sensitivity (88.8%; 203/228 cases). P. falciparum-infected sera revealed positive reactions (78.8%). The predominant immunoglobulin G (IgG) subclasses were segregated with IgG1 and IgG3.
doi:10.1128/CVI.00424-06
PMCID: PMC1828846  PMID: 17229883
15.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
16.  The enhancement of growth and differentiation of rat adrenal nerve cells by the addition of conditioned medium from human fibroblast cultures 
Cytotechnology  1998;26(2):125-130.
The growth of rat adrenal nerve cells was remarkably enhanced by supplementing the cultured medium from the human fibroblast cell line, Hs 68. Maximum specific growth rate and length of the neurites were observed as 0.076 (1/hr) and 0.026 mm, respectively in 20% supplement of five day old medium. In adding more than 20% of the cultured medium both cell and neurite growth was severely decreased. It was interesting that the cultured medium from Hs 68 cells could play a role in the extension of the neurites rather than in the growth of neurite cells. It was also found that molecules lower than 50,000 daltons in the conditioned medium could improve the growth of neurite bearing cells and the extension of the neurites than larger molecules. The efficacy of the proteins (<50,000 MW) was similar to that of human nerve growth factor and much better than that of basic fibroblast growth factor which was mainly secreted from human fibroblast cells.
doi:10.1023/A:1007942619463
PMCID: PMC3466676  PMID: 22358550
PC12 cell; differentiation; neurites; human fibroblast cells; basic fibroblast growth factor; nerve growth factor
17.  Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility 
Journal of Virology  1998;72(2):994-1004.
Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.
PMCID: PMC124570  PMID: 9444992
18.  Effect of HX108-CS supplementation on exercise capacity and lactate accumulation after high-intensity exercise 
Background
In the present study, we determined the effects of HX108-CS (mixed extract of Schisandra chinensis and Chaenomeles sinensis) supplementation on lactate accumulation and endurance capacity. Furthermore, we examined CK (creatine kinase), LDH (lactate dehydrogenase) activity to determine whether the HX108-CS affected markers of skeletal muscle injury in vivo and in vitro.
Methods
Exercise capacity was measured by an exhaustive swimming test using ICR mice divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, for the time-dependent measurements of blood lactate, CK, and LDH, Sprague–Dawley rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. High-intensity treadmill exercise was performed for 30 minutes. Blood samples were collected and analyzed during and after exercise. For the in vitro experiment, C2C12 cells were treated with HX108-CS to examine its effect on lactate production, CK, and LDH activity.
Results
Blood lactate concentration was significantly lowered immediately after treadmill exercise in HX108-CS group; however, there were no significant differences in activities of CK and LDH between HX108-CS and control during treadmill exercise and recovery phase. Furthermore, treatment with 100 mg/kg of HX108-CS led to a significant increase in the time to exhaustion in swimming test, and concurrently blood lactate concentration was significantly decreased in 50 and 100 mg/kg treated group. Moreover, our results of in vitro experiment showed that HX108-CS suppressed lactate production, CK, and LDH activity in a dose-dependent manner.
Conclusions
These results suggest that supplementation with HX108-CS may enhance exercise capacity by lowering lactate accumulation. This may in part be related to an amelioration of skeletal muscle injury.
doi:10.1186/1550-2783-10-21
PMCID: PMC3659049  PMID: 23587302
HX108-CS; Schisandra chinensis; Chaenomeles sinensis; Endurance capacity; Exercise capacity; Lactate accumulation

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