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Saprolegnia parasitica Isolated from Rainbow Trout in Korea: Characterization, Anti-Saprolegnia Activity and Host Pathogen Interaction in Zebrafish Disease Model

  • Shin, Sangyeop;Kulatunga, D.C.M.;Dananjaya, S.H.S.;Nikapitiya, Chamilani;Lee, Jehee;De Zoysa, Mahanama
    • Mycobiology
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    • v.45 no.4
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    • pp.297-311
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    • 2017
  • Saprolegniasis is one of the most devastating oomycete diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated oomycete belongs to the member of S. parasitica, supported by its typical features including cotton-like mycelium, zoospores and phylogenetic analysis with internal transcribed spacer region. Pathogenicity of isolated S. parasitica was developed in embryo, juvenile, and adult zebrafish as a disease model. Host-pathogen interaction in adult zebrafish was investigated at transcriptional level. Upon infection with S. parasitica, pathogen/antigen recognition and signaling (TLR2, TLR4b, TLR5b, NOD1, and major histocompatibility complex class I), pro/anti-inflammatory cytokines (interleukin $[IL]-1{\beta}$, tumor necrosis factor ${\alpha}$, IL-6, IL-8, interferon ${\gamma}$, IL-12, and IL-10), matrix metalloproteinase (MMP9 and MMP13), cell surface molecules ($CD8^+$ and $CD4^+$) and antioxidant enzymes (superoxide dismutase, catalase) related genes were differentially modulated at 3- and 12-hr post infection. As an anti-Saprolegnia agent, plant based lawsone was applied to investigate on the susceptibility of S. parasitica showing the minimum inhibitory concentration and percentage inhibition of radial growth as $200{\mu}g/mL$ and 31.8%, respectively. Moreover, natural lawsone changed the membrane permeability of S. parasitica mycelium and caused irreversible damage and disintegration to the cellular membranes of S. parasitica. Transcriptional responses of the genes of S. parasitica mycelium exposed to lawsone were altered, indicating that lawsone could be a potential anti-S. parasitica agent for controlling S. parasitica infection.

Protease Activity of Lactic Acid Bacteria Isolated from Korean Traditional Fermented Food (전통 발효식품으로부터 Protease 활성을 보유한 유산균의 분리 및 동정)

  • Kook, Moo Chang;Cho, Seok Cheol;Park, Hoon;Kim, Seung Seop;Pyun, Yu Ryang;Choi, Woon Yong;Lee, Hyeon Yong
    • Food Engineering Progress
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    • v.15 no.2
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    • pp.182-187
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    • 2011
  • A proteolytic lactic acid bacterium was isolated from Korean traditional fermented foods. The isolate BV-26, which had a protease activity (24 U/mg-crude protein), was identified as Lactobacillus plantarum by the API 50CHL kit and 16S rDNA analysis (99.9% of homology), and named as L. plantarum BV-26. Cell growth and protease activity of L. plantarum BV-26 was determined in MRS broth using 5L jar fermentor at $30^{\circ}C$. The maximum growth of L. plantarum BV-26 was reached at 18 hr in MRS broth, while protease activity of BV-26 was detectable at 12 hr and the highest activity was obtained after 16 hr cultivation. Therefore, we expect that the proteolytic lactic acid bacteria, L. plantarum BV-26, may be used as a starter for the fermentation of animal feed. Especially, the fermentation of soybean meal with the strain can be applied for improving feed utilization.

Effects of the Molecular Weight and Type of Chitosans on Shelf-life of Makkulli (막걸리의 저장성에 미치는 분자량별 및 형태별 키토산의 영향)

  • Shin, A-Ga;Jung, Yoo-Kyung;Lee, Ye-Kyung;Kang, Meung-Soo;No, Hong-Kyoon;Kim, Soon-Dong
    • Journal of Marine Bioscience and Biotechnology
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    • v.1 no.4
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    • pp.282-291
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    • 2006
  • Effects of the molecular weight and type of chitosans on shelf-life of Makkulli were evaluated during 18 days of storage at $25^{\circ}C$. Two types of chitosans were studied: ${\alpha}$-chitosans with 11 different molecular weights (water-soluble, Mw = 1, 8, 22, 43, 67 and 616 kDa; acid-soluble, Mw = 282, 440, 746, 1,110 and 2,025 kDa) and ${\beta}$-chitosan (acid-soluble, Mw = 577 kDa). Acid-soluble chitosans were applied as a form of chitosan-ascorbate. All chitosans were added to Makkulli at 0.002% concentration, the optimum concentration established in a preliminary test. Among 12 chitosans, the ${\alpha}$-chitosans with 22 and 440 kDa exhibited stronger antimicrobial effects than did other ${\alpha}$- and ${\beta}$-chitosans. The results for pH, acidity, alcohol concentration, viable cell counts, and sensory evaluation suggested that addition of ${\alpha}$-chitosans with 22 and 440 kDa increased the shelf-life of Makkulli by almost 1 week at $25^{\circ}C$ compared with that of control (without chitosan) and other chitosan-added groups. Extension of Makkulli shelf-life by 1 week is fairly significant in view of the magnitude of the total amount of Makkulli produced in Korea.

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Bioleaching of Mn(II) from Manganese Nodules by Bacillus sp. MR2 (Bacillus sp. MR2에 의한 망간단괴의 생물용출)

  • Choi, Sung-Chan;Lee, Ga-Hwa;Lee, Hong-Keum
    • Korean Journal of Microbiology
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    • v.45 no.4
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    • pp.411-415
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    • 2009
  • Some microorganisms are capable of leaching Mn(II) from nonsulfidic manganese ores indirectly via nonenzymatic processes. Such reductive dissolution requires organic substrates, such as glucose, sucrose, or galactose, as a source of carbon and energy for microbial growth. This study investigated characteristics of Mn(II) leaching from manganese nodules by using heterotrophic Bacillus sp. strain MR2 provided with corn starch as a less-expensive substrate. Leaching of Mn(II) at 25.6 g Mn(II) $kg^{-1}$ nodule $day^{-1}$ was accompanied with cell growth, but part of the produced Mn(II) re-adsorbed onto residual $MnO_2$ particles after 24 h. Direct contact of cells to manganese nodule was not necessary as a separation between them with a dialysis tube produced similar amount [24.6 g Mn(II) $kg^{-1}$ nodule $day^{-1}$]. These results indicated an involvement of extracellular diffusible compound(s) during Mn(II) leaching by strain MR2. In order to optimize a leaching process we tested factors that influence the reaction, and the most efficient conditions were $25\sim35^{\circ}C$, pH 5~7, inoculum density of 1.5~2.5% (v/v), pulp density of 2~3 g/L, and particle size <75 ${\mu}m$. Although Mn(II) leaching was enhanced as particle size decrease, we suggest <212 ${\mu}m$ as a proper size range since more grinding means more energy consumption The results would help for the improvement of bioleaching of manganese nodule as a less expensive, energy-efficient, and environment-friendly technology as compared to the existing physicochemical metal recovery technologies.

Predicting Factors of Roseola Infantum Infected with Human Herpesvirus 6 from Urinary Tract Infection

  • Ko, Hong-Ryul;Shin, Son Moon;Park, Sung Won
    • Childhood Kidney Diseases
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    • v.20 no.2
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    • pp.69-73
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    • 2016
  • Purpose: The aim of this study was to compare the clinical and laboratory features of infants with roseola infantum due to human herpesvirus 6 (HHV6) infection and those with urinary tract infection (UTI). Methods: We retrospectively reviewed the medical records of children who were hospitalized at Cheil General Hospital and Women's Health Care Center, College of Medicine, Dankook University, and diagnosed as having HHV6 infection or UTI. Results: Among the infants admitted between September 2014 and May 2016, 92 (male, 45 and female, 47) were included in the study and divided into a HHV6 infection group (n=50) and a UTI group (n=42). The relative risk of UTI compared with that of HHV6 infection increased with pyuria (P<0.001), increased with leukocytosis (mean white blood cell [WBC] count, $15,048{\pm}5,756/mm^3$ vs $87,916{\pm}54,056/mm^3$; P<0.001), increased with C-reactive protein (CRP) level ($4.89{\pm}4.85 mg/dL$ vs $1.04{\pm}1.76mg/dL$; P<0.001), and younger age ($6.3{\pm}3.2months$ vs $18.3{\pm}12.6months$; P<0.001). The relative risk of HHV6 infection compared with that of UTI increased with fever duration ($4.3{\pm}1.7days$ vs $2.8{\pm}1.7days$; P<0.001) and decreased with platelet (PLT) count ($373{\pm}94{\times}10^3/mm^3$ vs $229{\pm}90{\times}10^3/mm^3$; P<0.001). No significant differences were found between the HHV6 groups according to the presence or absence of pyuria. Conclusion: Pyuria, age, fever duration, WBC count, CRP level, and PLT count were the differentiating factors of HHV6 infection from UTI. However, sterile pyuria can occur in children with HHV6 infection. In the presence of pyuria, CRP level and PLT count were the strong predictors of UTI compared with HHV6.

Establishment and Characterization of Multipotent Germ Line Stem Cells (MGSCs) from Neonatal Mouse Testis (신생 생쥐 고환에서 기인한 다분화능 생식줄기세포주의 확립 및 특성 분석)

  • Han, Sang-Chul;Song, Haeng-Seok;Jun, Jin-Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.35 no.1
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    • pp.39-48
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    • 2008
  • Objective: The aim of this study was to investigate whether multipotent germline stem cells (MGSCs) can be established from neonatal mouse testis. Methods: Various cells containing MGSCs were collected from neonatal testis of ICR mice and allocated to plates for in vitro culture. After 7 days in culture, the cells were passed to a fresh culture plate and continuously cultured. From the third or fourth passage, the presumed MGSCs were cultured and maintained on mitomycin C-inactivated STO feeder cells. The MGSCs were cultured in a condition where mouse embryonic stem cells (ESCs) are cultured. Characteristics of the MGSCs were evaluated by RT-PCR, immunocytochemistry, alkaline phosphatase activity, karyotyping, and transmission electron microscopy. Results: Two MGSCs lines were established from 9 pooled sets of neonatal testicular cells. MGSCs colonies were morphologically undistinguishable from ESCs colonies and both MGSC lines as well as ESCs expressed undifferentiated stem cell markers, such as Thy-1, Oct-4, Nanog, Sox2 and alkaline phosphatase. Fine structure of undifferentiated MGSCs were similar to those of ESCs and 60% of MGSCs (12/20) had normal karyotype at passage 10. They were able to form embryoid bodies (EBs) and MGSC-derived EBs expressed marker genes of three germ layers. Conclusion: We could establish the MGSCs from neonatal mouse testis and they were differentiated to multipotent lineages of three germ layers. Molecular characteristics of MGSCs were similar to those of ESCs. Our results suggest a possibility that multipotent stem cells derived from testis, the MGSCs, could replace the ESCs in biotechnology and regenerative medicine.

Effects of Danggwisayeokgaohsuyusaenggang-tang(當歸四逆加吳茱萸生薑湯) on Collagen-induced Arthritis in Mice (Collagen으로 유발된 생쥐의 관절염에 대한 당귀사역가오수유생강탕(當歸四逆加吳茱萸生薑湯)의 효과)

  • Kim, Min-Kyun;Oh, Min-Seok
    • Journal of Korean Medicine Rehabilitation
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    • v.21 no.2
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    • pp.63-85
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    • 2011
  • Objectives : This study was carried out to know the effects of Danggwisayeokgaohsuyusaenggang-tang(hereinafter referred to DST) on arthritis induced by collagen on DBA/1 OlaHsd mice. Methods : For this purpose, DST was orally administered to mouse with arthritis induced by collagen II. Cytotoxicity, high performance liquid chromatograph(HPLC) analysis, arthritis index, value of immunocyte in draining lymph node and paw joint, cytokine were measured in vivo. Results : 1. The cytotoxicity against human fibroblast cells(hFCs) was not measured in any concentration. 2. In HPLC analysis, There are high peak patterns at 8 minute(min), 12 min, 35 min, 45 min. 3. The arthritis index was decreased significantly. 4. The degree of arthritis induced damage of joint of DST group is slight compared with control group in histopathologic observation(Hematoxylin and eosin stain(H&E), Masson's trichrome(M-T) staining). 5. In total cell counts of draining lymph node(DLN) and paw joint, the cells in DLN decreased significantly on DST 200 mg/kg and the cells in paw joint decreased significantly on 200 mg/kg and 50 mg/kg. 6. In DLN, $CD4^+/CD25^+$, $CD3^+/CD69^+$, major histocompatibility complex(MHC), class-II/$CD11c^+$ cells decreased significantly on DST 200 mg/kg and 50 mg/kg $CD3^+/CD8^+$ cells decreased significantly on DST 200 200 mg/kg, $CD4^+$, $CD3^+/CD44^+$ cells decreased. 7. In paw joints, $CD4^+$, $CD11b^+/Gr-1^+$ cells decreased significantly on DST 200 mg/kg and 50 mg/kg. 8. In joints, levels of $IL-1{\beta}$, IL-6, $TNF-{\alpha}$, cyclo-oxygenase-2(COX-2), NOS-II were decreased on DST 200 mg/kg and DST 50 mg/kg. 9. In analysing of cytokine in CD3/CD28 activated spleen, IL-17 was decreased significantly, IL-4 was increased significantly $INF-{\gamma}$ was decreased on DST 200 mg/kg. 10. In analysing of cytokine in collagen activated spleen, IL-17 were decreased significantly, IL-4 was increased significantly. Conclusions : This results demonstrated that DST suppressed the inflammatory progression of collagen-induced arthritis(CIA) mice and supported further studies are required to survey continuously in looking for the effective substance and mechanism in the future.

Second allogeneic hematopoietic stem cell transplantation in children to overcome graft failure or relapse after initial transplant (조혈모세포이식 후 생착 실패나 재발한 소아환자에서 2차 이식의 의의)

  • Kim, Dong-Yeon;Kim, Do Kyun;Kim, Soo Young;Kim, Seok Joo;Han, Dong Gyun;Baek, Hee Jo;Kook, Hoon;Hwang, Tai-Ju
    • Clinical and Experimental Pediatrics
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    • v.49 no.12
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    • pp.1329-1339
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    • 2006
  • Purpose : Failure of hematopoietic stem cell transplantation(HSCT) may be encountered in practice because of either relapse of the malignancy or dysfunction of the graft. Second HSCT may be the only option for some patients whose initial HSCT failed. Methods : From May, 1991 to December, 2004, 115 HSCTs were performed at the Pediatric Blood & Marrow Transplantation Center, Chonnam National University. This study was a retrospective analysis of the medical records of 15 patients who received the second HSCT after initial graft. Results : Among eight patients with nonmalignant diseases, two patients underwent the second HSCT because of primary graft failure and five because of late graft rejection. The remaining Fanconi anemia patient was re-transplanted due to development of AML. Two patients died and one experienced primary graft failure, but is still alive. The Kaplan-Meier 5-year overall survival rate was 75 percent and the disease free survival rate was 62.5 percent in nonmalignant diseases. All malignant patients underwent second transplants because of relapses. Four died of relapse and one of treatment-related complications. The Kaplan-Meier 2-year overall and event free survival rate was 28.6 percent each in malignant diseases. Conclusion : Second HSCT for graft dysfunction of nonmalignant disease seems to be feasible and should be considered as a standard practice. The relapse of malignant diseases remains a big obstacle even after the second HSCT, although a small portion of patients might be salvaged. Further investigation of novel therapeutic strategies, as well an the understanding of the biology should be explored.

Seasonal Changes of Species Composition and Standing Crop of Phytoplankton in the Ark Shell Scapharca broughtonii Farming Areas of Jinhae Bay (진해만 피조개 Scapharca broughtonii 양식장 식물플랑크톤의 종조성과 현존량의 월별변화)

  • Kim, Byoung-Hak;Min, Byeong-Hee;Choi, Nak-Joong;Oh, Bong-Se;Park, Ki-Yeol;Min, Kwang-Sik
    • Journal of Aquaculture
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    • v.21 no.3
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    • pp.157-166
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    • 2008
  • Species composition and standing crop of phytoplankton were investigated in the ark shell Scapharca broughtonii farming areas from March, 2006 to February, 2007 in Jinhae Bay. Water temperature ranged from 7.56 to $25.90^{\circ}C$, salinity from 13.74 to 34.78 psu, dissolved oxygen from 4.13 to 13.20 mg/L, chlorophyll $\alpha$ from 2.77 to 104.98 $mg/m^2$ and pH from 7.83 to 8.65. Dominant species was Nitzschia and Rhizosolenia from March to May, Skeletonama costatum and Prorocentrum from June to August, Skeletonama costatum, Thalassiosira, Chaetoceros from September to November and Thalassiosira, Chaetoceros from December to February. Colonial diatoms were more dominant than the single cell diatoms. Standing crop was the highest in July at three stations. Standing crop of Skeletonama costatum was the highest as 1,760.0 cells/mL at St. 1, 1,075.2 cells/mL at St. 2 and 698.4 cells/mL at St. 3 in July.

Cloning of a Glutathione S-Transferase Decreasing During Differentiation of HL60 Cell Line (HL6O 세포주의 분화 시 감소 특성을 보이는 Glutathione S-Transferase의 클로닝)

  • Kim Jae Chul;Park In Kyu;Lee Kyu Bo;Sohn Sang Kyun;Kim Moo Kyu;Kim Jung Chul
    • Radiation Oncology Journal
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    • v.17 no.2
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    • pp.151-157
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    • 1999
  • Purpose : By sequencing the Erpressed Sequence Tags of human 걸ermal papilla CDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL6O cell line. Materials and Methods : K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Nothern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusicn expression system and the protein product was identified on SDS-PAGE. Results : K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares $70\%$, identity with that of rat glutathione 5-transferase kappa 1 (rGSTKl). The transcripts were expressed in a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in coloreetal cancer and melanoma cell lines. Conclusion : Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.

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