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Comparative Diagnostic Studies on Serologic and Molecular Biological Tests Against Haemorrhagic Fever with Renal Syndrome (신증후출혈열 환자의 혈청학적 및 분자생물학적 진단 검사법 비교)

  • 우영대;문희주;배형준
    • Biomedical Science Letters
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    • v.6 no.2
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    • pp.141-149
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    • 2000
  • The etiologic agents of haemorrhagic fever with renal syndrome (HFRS) in Korea are Hantaan and Seoul virus in the genus Hantavirus, family Bunyaviridae. Antibody titers of sera from HFRS patients against Hantaan virus were measured by immunofluorescent antibody technique (IFAT), enzyme-linked immunosorbent assay (ELISA), high density composite particle agglutination (HDPA) and plaque reduction neutralization test (PRNI). PRNT and nested reverse transcriptase polymerase chain reaction (nested RT-PCR) was used for serotypic differentiation of Hantaviruses against Hantaan and Seoul virus. Eight doubtful HFRS patients showed higher fluorescent, IgG ELISA, agglutination and neutralizing antibody titer by IFAT, ELISA IgG, HDPA and PRNT, respectively Five out of them showed high IgM antibody titer by IgM capture ELISA against Hantaan virus, remarkably. Fifteen HFRS patients showed higher fluorescent antibody titer by IFAT. In PRNT, 12 out of them showed high neutralizing antibody titer against HTNV, 2 against SEOV and 1 against both viruses. In nested RT-PCR using serotype specific-primer, 3 out of them showed positive against HTNV and 1 against SEOV.

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Overexpressed Derlin-1 Inhibits ER Expansion in the Endothelial Cells Derived from Human Hepatic Cavernous Hemangioma

  • Hu, Dong;Ran, Yu-Liang;Zhong, Xing;Hu, Hai;Yu, Long;Lou, Jin-Ning;Sun, Li-Xing;Yang, Zhi-Hua
    • BMB Reports
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    • v.39 no.6
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    • pp.677-685
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    • 2006
  • Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be targeted for refolding or degradation to maintain the homeostasis of the ER. Derlin-1 was reportedly implicated in the retro-translocation of misfolded proteins from the ER to the cytosol for degradation. In this report, we showed that Derlin-1 was down-regulated in the endothelial cells derived from human hepatic cavernous hemangioma (CHEC) compared with other tested cells. Electron microscopy analysis showed that ER was aberrantly enlarged in CHEC cells, but not in other tested cells. When overexpressed, Derlin-1 induced the dilated ER to return normal size. This ER dynamic was associated with the activation of unfolded protein response (UPR). In CHEC cells where Derlin-1 was down-regulated, increased expression of the immunoglobulin heavy chain-binding protein (Bip) and UPR-specific splicing of X-box DNA-binding protein 1 (XBP1) mRNA were detected, as compared with that in other tested cells, indicating that UPR was activated. After Derlin-1 overexpression, the extent of UPR activation diminished, as evidenced by decreased expression of Bip, reduced amount of the spliced form of XBP1 ($XBP1_S$), and elevated expression of the unspliced form of XBP1 ($XBP1_U$). Taken together, these findings provide another example of a single protein being able to affect ER dynamic in mammalian cells, and an insight into the possible molecular mechanism(s).

KBTBD7, a novel human BTB-kelch protein, activates transcriptional activities of SRE and AP-1

  • Hu, Junjian;Yuan, Wuzhou;Tang, Ming;Wang, Yuequn;Fan, Xiongwei;Mo, Xiaoyang;Li, Yongqing;Ying, Zaochu;Wan, Yongqi;Ocorr, Karen;Bodmer, Rolf;Deng, Yun;Wu, Xiushan
    • BMB Reports
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    • v.43 no.1
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    • pp.17-22
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    • 2010
  • In this study, a novel member of BTB-kelch proteins, named KBTBD7, was cloned from a human embryonic heart cDNA library. The cDNA of KBTBD7 is 3,008 bp long and encodes a protein product of 684 amino acids (77.2 kD). This protein is highly conserved in evolution across different species. Western blot analysis indicates that a 77 kD protein specific for KBTBD7 is wildly expressed in all embryonic tissues examined. In COS-7 cells, KBTBD7 proteins are localized to the cytoplasm. KBTBD7 is a transcription activator when fused to GAL4 DNA-binding domain. Deletion analysis indicates that the BTB domain and kelch repeat motif are main regions for transcriptional activation. Overexpression of KBTBD7 in MCF-7 cells activates the transcriptional activities of activator protein-1 (AP-1) and serum response element (SRE), which can be relieved by siRNA. These results suggest that KBTBD7 proteins may act as a new transcriptional activator in mitogen-activated protein kinase (MAPK) signaling.

The Effects of Hantaan Virus on the Expression of Platelet Activating Factor Receptor and on the Activity of Platelet Activating Factor Acetylhydrolase (한탄바이러스가 혈소판활성인자 수용체 발현 및 혈소판활성인자 분해효소 활성에 미치는 영향)

  • Hwang, Ji-Young;Park, Jong-Won;Hong, Sae-Yong;Park, Ho-Sun
    • Journal of Yeungnam Medical Science
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    • v.25 no.1
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    • pp.41-49
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    • 2008
  • Background : The central physiological derangement of hemorrhagic fever with renal syndrome (HFRS) caused by hantaan virus (HTNV) is a vascular dysfunction, manifested by hemorrhage, impaired vascular tone and increased vascular permeability. Platelet activating factor (PAF), whose actions are mediated through a specific receptor, is a potent bioactive lipid. PAF has diverse biological functions in the vascular system, such as increasing vascular permeability, adhesion of leukocytes to the endothelium and reduction of cardiac output, which result in hypotension and shock. The goal of the present study was to investigate whether PAF is involved in the pathogenesis of HFRS. For this purpose, we evaluated the effect of HTNV on the expression of PAF receptor (PAF-R) and on the activity of PAF-acetylhydrolase (PAF-AH) instead of PAF because PAF is rapidly degraded by PAF-AH in vivo. Materials and methods : To evaluate the expression of PAF-R, we performed reverse-transcription PCR, western blot and FACS analyses using HTNV-infected human umbilical vein endothelial cells (HUVECs) and non-infected (control) HUVECs. In addition, we measured the activity of plasma PAF-AH in HFRS patients and normal healthy persons. Results : The mRNA and protein expression of PAF-R was increased in HTNV-infected HUVECs compared with control HUVECs at 2 and 3 days post-infection (d.p.i.). FACS analysis showed that HTNV induced the surface expression of PAF-R in HUVECs from 2 d.p.i. The activity of plasma PAF-AH was 2.5-fold lower in HFRS patients than in normal healthy persons. Conclusion : Increased PAF-R expression by HTNV might increase the responsiveness to PAF in endothelial cells. Reduced PAF-AH activity in the blood of HFRS patients might delay PAF degradation. These results suggest that changes in PAF-R and PAF-AH by HTNV might influence to PAF activity and might be involved in the vascular dysfunction of HFRS.

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Gluconacetobacter persimmonis sp. nov., Isolated from Korean Traditional Persimmon Vinegar

  • Yeo, Soo-Hwan;Lee, Oh-Seuk;Lee, In-Seon;Kim, Hyun-Soo;Yu, Tae-Shick;Jeong, Yong-Jin
    • Journal of Microbiology and Biotechnology
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    • v.14 no.2
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    • pp.276-283
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    • 2004
  • Screening was performed to isolate cellulose-producing microorganisms from the Korean traditional fermented persimmon vinegar. The resulting strain, KJ $145^{T}$, was then taxonomically investigated by phenotypic characterization, particularly chemotaxonomic, and by phylogenetic inference based on a 16S rDNA sequence analysis including other related taxa. Strain KJ $145^{T}$ was found to grow rapidly and form pale white colonies with smooth to rough surfaces on a GYC agar. Strain KJ $145^T$ also produced acetate from ethanol, and was tolerable to 10% ethanol in SM medium. In a static culture, a thick cellulose pellicle was produced, and in GYC broth, the strain grew at temperatures ranging from 28 to $40^\circ{C}$ with an optimum pH of 4.0. The genomic DNA G+C content of strain KJ $145^T$ was 61.9 mol%, and the predominant ubiquinone was Q 10 as the major quinone and Q9 as the minor quinone. The major cellular fatty acids were $C_{16:0}$ and the sum in feature 7 ($C_{18:1}$ w9c, w12t and/or w7c). A 16S rRNA-targeted oligonucleotide probe specific for strain KJ $145^T$was constructed, and the phylogenetic position of the new species was derived from a 16S rDNA-based tree. When comparing the 16S rDNA nucleotide sequences, strain KJ $145^T$ was found to be most closely related to G. hansenii LMG $1527^T$ (99.2%), although KJ $145^T$ was still distinct from G. hansenii LMG $l527^T$ and G. xylinus LMG $1515^T$ in certain phenotypic characteristics. Therefore, on the basis of 16S rDNA sequences and taxonomic characteristics, it is proposed that strain KJ $145^T$ should be placed in the genus Gluconacetobacter as a new species, Gluconacetobacter persimmonis sp. nov., under the type-strain KJ $145^T$ (=KCTC =$10175BP^T$=KCCM=$10354^T$).

The Phylogenetic Analysis of the NS-5 Region Sequence of Hepatitis G Viruses Isolated in Korea (국내에서 분리된 G형 간염바이러스 NS-5 Region 염기서열의 계통학적 분석)

  • Jee, Young-Mee;Kim, Ki-Soon;Cheon, Doo-Sung;Park, Jeong-Koo;Kang, Young-Hwa;Lee, Yoon-Sung;Chung, Yoon-Suk;Kim, Ji-Eun;Yoon, Jae-Deuk
    • The Journal of Korean Society of Virology
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    • v.29 no.1
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    • pp.45-53
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    • 1999
  • We examined the hepatitis G virus infections among 227 Koreans who were healthy or were suspected of hepatitis and determined the phylogenetic relationship based on a part of the NS-5 region of 5 positive samples. Viral RNA was extracted from sera and cDNA was synthesized and subsequently amplified by RT-PCR (reverse transcription-polymerase chain reaction) or RT-nested PCR using random hexamer and NS-5 specific primers (470-20-1-77F, 470-20-1-211R, HGVNESTFO, HGVNESTRE). Five positives were found to belong to samples of patients showing symptoms of viral hepatitis. Primers used for PCR or nested PCR were derived from the NS-5 region. On the other hand, no amplification was detected using primers derived from the 5'-NCR (G-146F, G-401R). We performed TA cloning and sequencing of 5 amplified fragments, and their sequences were compared with those of foreign isolates of HGV. The phylogenetic analysis using MegAlign programme of DNAstar has shown that the Korean isolates are clustered on the phylogenetic tree. In summary, we confirmed the hepatitis G virus infection in 5 cases out of 12 patients showing the symptoms of viral hepatitis. The phylogenetic analysis of sequences of 5 amplified fragments showed that their relations to each other were closer than those to the foreign HGV isolates reported.

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Membranous Nephropathy Associated with Epstein-Barr Virus Infection in a Child (소아에서 엡스타인-바 바이러스 감염과 관련한 막성 신병증 1례)

  • Lee, Eun-Hee;Lim, Dong-Hee;Yim, Hyung-Eun;Yoo, Kee-Hwan;Won, Nam-Hee;Hong, Young-Sook;Lee, Joo-Won
    • Childhood Kidney Diseases
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    • v.12 no.1
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    • pp.88-92
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    • 2008
  • Infection of Epstein-Barr virus(EBV) gives rise to a broad spectrum of clinical manifestations in children. Although renal involvement is rare, diverse renal manifestations are known from hematuria to acute renal failure. Secondary membranous nephropathy(MN) associated with systemic EBV infection is an uncommon renal pathology and only two cases have been reported. We are adding another case of MN associated with EBV infection in a child. An 8-year-old girl was admitted for renal biopsy. She had been followed up for microscopic hematuria and intermittent proteinuria for 5 months. There had been no specific findings in serology and radiology. Tonsil biopsy had been done due to exudative tonsillar hypertrophy and enlarged multiple cervical lymph nodes. And it showed EBV-associated lymphoproliferative findings. Serologic tests for EBV showed positive evidence of recent infection; viral capsid antigen(VCA) IgM was borderline positive, VCA IgG and early antigen IgG were positive, and EB nuclear antigen IgG was negative. In Situ Hybridization of tonsil for EBV mRNA was positive. Because her proteinuria and hematuria were aggravated at that time(protein 3 +, RBC >60/HPF), renal biopsy was done. Renal biopsy showed the findings of MN, characterized by thickened capillary walls with epimembranous spikes on light microscopy and subepithelial, mesangial and subendothelial electron dense deposits on electron microscopy. On immunofluorescence microscopy, IgG, C1q, kappa and lambda chains were positive. After steroid administration, proteinuria and hematuria resolved gradually within 6 months.

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Characterization of Prunus necrotic ringspot virus Isolate from Peach in Korea (국내 복숭아에서 분리한 Prunus necrotic ringspot virus의 특성)

  • Kim, Hyun-Ran;Lee, Sin-Ho;Shin, Il-Sheob;Kim, Jeong-Hee;Cho, Kang-Hee;Heo, Seong;Kim, Jeong-Soo;Choi, Yong-Mun
    • Research in Plant Disease
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    • v.15 no.3
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    • pp.170-174
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    • 2009
  • In this paper, we report a characterization of Prunus necrotic ringspot virus (PNRSV) isolate. The virus was identified from 'Yumyeong' peach showing mild mosaic on leaves in commercial orchard of 'Umsung', Chungbuk province in Korea. The virus isolate produced ringspot symptom on the inoculated cotyledons and systemic mosaic and malformation on the upper leaves of Cucumis sativus. Systemic mottles were appeared in Chenopodium quinoa. When the buds of the virus infected stem were grafted on the healthy young Prunus persica GF305 seedlings, line pattern with mosaic appeared within 3 months. Isometric virus-like particles were found in parenchyma cells and plasmodesmata of C. sativus leaves inoculated mechanically with the virus. The cDNA fragments of PNRSV coat protein (CP) region, approximately 675bp, were synthesized from genomic RNA extracted from virus-infected leaves by RT-PCR using specific primer pairs. Partial nucleotide sequences of the CP regions were determined and analyzed with the known PNRSV. The CP gene of PNRSVKorea isolates showed 93.9~94.7% similarity to the 4 known PNRSV isolates.

Cloning and Characterization of Dihydroflavonol 4-reductase (DFR) from Matthiola incana R. Br. (Stock(Matthiola incana R. Br.)으로부터 색소유전자의 분리 및 분석)

  • 민병환;김석원;오승철;유장렬
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.5
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    • pp.341-346
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    • 1998
  • In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol 4-reductase (DFR) in Matthiola incana R. Br. A heterologous cDNA probe from Zea mays was used to isolate full-size DFR cDNA clone from a corolla-specific cDNA library. Comparison of the coding region of this DFR cDNA sequence including the sequences of Zea mays, Anthirrinum majus, Petunia hybrida, Callistephus chinensis, Dianthus caryophyllus and Rosa hybrida reveals a identity higher than 61% at the nucleotide level. The DFR transcript is G/C rich in monocotyledonous plants show a strong codon bias preferring codons with a G or C in the third position. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants and by in vitro expression yielding an enzymatically active reductase. Genomic southern blot analysis showed the presence of one gene for DFR in Matthiola incana. Northern blot analysis of the DFR wild type and mutant lines showed that the lack of DFR activity in the stable acyanic mutant k17b is clearly by a transcriptional block of the DFR gene.

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Optimization of Indole-3-Acetic production by phosphate solubilization bacteria isolated from waste mushroom bed of Agaricus bisporus

  • Walpola, Buddhi Charana;Noh, Jae-Geun;Kim, Chan Kyem;Kyung, Ki-Cheon;Kong, Won-Sik;Yoon, Min-Ho
    • Journal of Mushroom
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    • v.11 no.2
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    • pp.53-62
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    • 2013
  • A total of 35 phosphate solubilizing bacterial strains were isolated from waste mushroom bed of Agaricus bisporus in Buyeo-Gun, Chungnam and screened for the production of indole acetic acid (IAA). The best IAA producing strain was identified as Pantoea rodasii using 16S rRNA analysis. In addition to the IAA production, this strain could act as an efficient phosphate solubilizer (1100 ${\mu}g$ $ml^{-1}$ after 5 days of incubation) also. The selected strain was cultured under different conditions in order to assess the optimum conditions for maximum IAA production. The nutrient broth (NB) medium was recorded as the best medium, where the maximum IAA production (229 ${\mu}g$ $ml^{-1}$) was recorded at the start of stationary phase (12 hours after inoculation) of the bacteria growth. The performance of the strain was found to be maximum at the temperature of $30^{\circ}C$ followed by $25^{\circ}C$. IAA production was found to be increased with increasing tryptophan concentration (from 0.1 to 0.6%), however beyond this limit, a slight reduction in IAA production was observed. The strains' ability to produce IAA was further confirmed by extraction of crude IAA and subsequent TLC analysis. A specific spot from the extracted IAA preparation was found corresponding with the standard spot of IAA with same $R_f$ value. The results of HPLC analysis conducted in identifying and quantifying the IAA production more precisely, are in agreement with the results of the assessment done with colorimetric method. As revealed by the results of the pot experiment, the isolated strain could significantly enhance the growth (as measured by shoot and root growth) of mung bean plants compared to that of non-inoculated plants. Therefore it can be concluded that the present strain, Pantoea rodasii has great potential to be used as bio-inoculants.