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Genomic Analysis of Human Papillomavirus Type 16 in Cervical Cancer.

Seong Ho TAE ; Yong Tae HAN ; Young Lae CHO ; Chul Hee CHYOI ; Hak Sun YU ; Sung Yong SEOL ; Dong Teak CHO ; Yoo Chul LEE

Journal of Bacteriology and Virology.2002;32(4):441-448.

To study the correlation of human papillomavirus (HPV) infection with clinical stage in cervical abnormalities, 17 cases of normal cervical tissue and 69 cases of abnormal cervical tissue (cervical intraepithelial neoplasia and invasive cervical cancer) was examined by PCR with HPV-specific consensus primers. One case (5.9%) of normal cervical tissue and 42 cases (60.9%) of abnormal cervical tissues harbored HPV. To investigate the integration of HPV genome in 24 cases of HPV 16 positive cervical cancer, E2 gene of HPV 16 was amplified. Integration of HPV 16 was found in 7 cases (29.2%) with E2 disruption. All samples with E2 disruption were from invasive cervical cancer. A multiplex PCR for the mapping of integrated HPV 16 genome with an anchor primer and indicator primers showed that 11 cases (45.8%) were disrupted somewhere in HPV genome but E6, E7, and LCR regions were conserved in all cases. Seven types of integrated HPV genome from long- (7,062 bp) to short-conserved type (3,204 bp) with various deletions were detected by the multiplex PCR. These results show that integration can be detected more accurately by multiplex PCR than by E2 PCR, and E2 disruption is not a critical event of integration
Consensus ; Genome ; Human papillomavirus 16 ; Humans* ; Multiplex Polymerase Chain Reaction ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms*

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Expression and Characterization of Human Immunodeficiency Virus Type 1 Envelope Mutant Glycoproteins by Using Baculovirus Expression System.

Yoon LEE ; Ji Yoon RYU ; Kil Soo LEE ; Yong Soo BAE ; Soo Young CHOI ; Jin Seu PARK

Journal of Bacteriology and Virology.2002;32(4):431-440.

Considerable effort has been directed at understanding the structure and function of HIV-1 envelope glycoproteins. It has been difficult to characterize HIV-1 envelope glycoproteins due to the limited availability of these proteins from virus particles or infected cells. To facilitate the structural and functional analysis of HIV-1 envelope glycoproteins, recombinant baculoviruses were generated to express wild type or mutant HIV-1 envelope glycoproteins. The gp160 precursor protein as well as the gp120 glycoprotein were detected in the cell infected with recombinant BacENVw.t containing wild type HIV-1 envelope gene. In the insect cells infected with recombinant BacENVc with mutations at the cleavage site of gp160, a precursor form of envelope glycoprotein was produced, but not secreted into the culture medium. However, the insect cells infected with recombinant BacENVc/t containing both mutations at the cleavage site and membrane spanning region produced mutant envelope glycoproteins that were efficiently secreted into the culture medium in the form of precursor. Therefore the recombinant HIV-1 envelope glycoproteins produced in this system would be useful as immunogens in the development of a vaccine against AIDS.
Baculoviridae* ; Glycoproteins* ; HIV* ; HIV-1* ; Humans* ; Insects ; Membranes ; Staphylococcal Protein A ; Virion

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Expression and Purification of Glycoprotein G1 of Hantaan Virus in E. coli System.

Kyu Ri CHOI ; Jae Hwan NAM ; Woo Young CHOI ; Young Ran JU ; Keun Yong PARK ; Hae Wol CHO

Journal of Bacteriology and Virology.2002;32(4):421-430.

Envelope glycoprotein 1 (G1) and glycoprotein 2 (G2) of Hantaan (HTN) virus are believed to be major viral antigens that can induce neutralizing immunity against HTN virus infection. The purpose of this study is to clone and express G1 gene in an E. coli expression system. The truncated G1 gene (amino acid residues 35 to 123) of the HTN virus strain 76-118 was amplified by polymerase chain reaction (PCR). The 0.28 kb PCR product was cloned into pCR2.1 vector and named as pCGS1. The truncated G1 gene was excised from the pCGS1 and subcloned into the BamHI and SalI sites of pGEX-4T-2 and named pGGS1. The nucleotide sequence of the 0.28 kb truncated G1 gene was determined. It is revealed four non-silent nucleotide substitutions between the published sequence of strain HTN virus strain 76-118 and our stock of HTN virus strain 76-118 (passaged several times in our laboratory). The first G1 mutation was found to constitute an A to G nucleotide substitution, giving raise to an asparagine to serine mutation at residue 64. The second G1 mutation was found to constitute an A to C nucleotide substitution, giving raise to an lysine to threonine mutation at residue 112. The third G1 mutation was found to constitute an A to C nucleotide substitution, giving raise to an lysine to threonine mutation at residue 112. The fourth G1 mutation was found to constitute an G to A nucleotide substitution, giving raise to an glutamic acid to lysine mutation at residue 117. The truncated G1 gene was expressed as a 37 kDa protein fused to glutathione-S-transferase (GST). The GST fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and reacted with the sera from patients of hemorrhage fever with renal syndrome (HFRS). One of 12 serum samples from HFRS patients was reactive with the 37 kDa fusion protein strongly. Three sera reacted moderately with the fusion protein. Six sera reacted only weakly with the protein, while remaing two were non-reactive. Control sera from patients with scrub typhus leptospirosis, or negative HFRS did not react with the recombinant fusion protein.
Antigens, Viral ; Asparagine ; Base Sequence ; Blotting, Western ; Chromatography, Affinity ; Clone Cells ; Fever ; Glutamic Acid ; Glutathione ; Glycoproteins* ; Hantaan virus* ; Hemorrhage ; Hemorrhagic Fever with Renal Syndrome ; Humans ; Leptospirosis ; Lysine ; Polymerase Chain Reaction ; Scrub Typhus ; Sepharose ; Serine ; Threonine

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Concurrent Impairment of Th1 and Th2 Response in Thermal Injury.

Jeong Su DO ; Young Hyun KIM ; Sang Yun NAM

Journal of Bacteriology and Virology.2002;32(4):411-420.

Large numbers of reports have shown that thermal injury (TI) causes a wide spectrum of defects in immune response that lead to a high susceptibility to various opportunistic infections. However, it is still a matter of debate whether TI induces Th2 polarization or global impairment in Th1/Th2 response. In this study, TI in a mouse model was induced by exposing shaved dorsal skin to boiling water and cytokine production was analyzed. At day 2 of injury, whole spleen cells and T cells were collected and then stimulated with an anti-CD3 antibody. The levels of cytokine secretion were determined by cytokine ELISA. Production of IFNgamma and IL 4 by whole spleen cells from injured mice were concurrently decreased when compared to those from sham-injured controls. Proportional changes in T, B, and T-subset cells were not accompanied. Using purified T cells devoid of accessory cells (AC), it was shown that those defects resulted primarily from lowered T cell potentials. By using mixed cultures of sham T and TI-AC and vice versa, it was revealed that AC also acted as inhibitor cells in IFNgamma and IL 4 production in less extent. Blockade of glucocorticoid signals rendered the T cells partially resistant to TI-induced inhibition in IFNgamma and but not IL 4 production. These results clearly demonstrate that TI induces overall suppression in Th1 and Th2 response through T cell dysfunction together with the inhibition of AC activity, and that reduction in only IFNgamma but not IL 4, production may be caused, in part, by corticosteroid hormone that is secreted prominently during trauma.
Animals ; Enzyme-Linked Immunosorbent Assay ; Mice ; Opportunistic Infections ; Skin ; Spleen ; T-Lymphocytes ; Water

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Identification and Functional Characterization of Differentially Expressed Genes in Human-derived Monocytic Cell Line U937 Infected with Mycobacterium tuberculosis H37Rv and Mycobacterium marinum: Comparative Evaluation of IL-8.

Chang Hwa SONG ; Ji Sook LEE ; Hwa Jung KIM ; Jeong Kyu PARK ; Tae Hyun PAIK ; Eun Kyeong JO

Journal of Bacteriology and Virology.2002;32(4):401-410.

Although Mycobacterium marinum is closely related to M. tuberculosis H37Rv (M. tbc) genomically, clinical outcome of human infection is quite different. The role of the host macrophage in determining differential pathologic responses was analyzed using an in vitro model of macrophage infection. By using subtractive hybridization, thirty-two differentially expressed genes were identified in the monocytic cell line U937 infected with M. tbc or M. marinum. Among them, IL-8 mRNA expression was more prominent in the M. tbc-infected U937 cells by Northern hybridization than in those infected with M. marinum. The IL-8 production was significantly lower in M. marinum-infected U937 or monocytes when compared with those infected by other strains of mycobacteria, such as M. tuberculosis H37Ra, M. bovis BCG or M. smegmatis. To identify possible mechanisms underlying these differences, changes in the expression of molecules such as nuclear factor-kappaB (NF-kappaB) involved in the signaling pathway activated by mycobacteria were assessed. U937 cells infected with M. tbc showed a significant degradation of IkBa proteins compared with M. marinum-infected U937 cells. Collectively, these results implicate distinct differences in IL-8 production in human macrophages infected with M. tbc or M. marinum, and suggest important role of IL-8 in the immunopathogenesis of tuberculosis.
Cell Line* ; Humans ; Interleukin-8* ; Macrophages ; Monocytes ; Mycobacterium bovis ; Mycobacterium marinum* ; Mycobacterium tuberculosis* ; Mycobacterium* ; RNA, Messenger ; Tuberculosis ; U937 Cells

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Depression of Both Interferon-gamma and Tumor Necrosis Factor-alpha Production by Peripheral Blood Mononuclear Cells from Chronic Refractory Tuberculosis Patients.

Ji Sook LEE ; So Hyun LEE ; Chang Hwa SONG ; Jae Hyun LIM ; Hwa Jung KIM ; Jeong Kyu PARK ; Tae Hyun PAIK ; Chul Hee KIM ; Suck Jun KONG ; Mal Hyun SHON ; Eun Kyeong JO

Journal of Bacteriology and Virology.2002;32(4):393-400.

Understanding human immune responses in chronic refractory tuberculosis (CRTB) is important for developing immunotherapy against the disease. The aim of this study was to examine cytokine responses [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-10] by peripheral blood mononuclear cells (PBMCs) in CRTB patients after in vitro stimulation with the 30-kDa or purified protein derivative (PPD) antigen (Ag). Most of the CRTB cases were multidrug-resistant (MDR) TB. The results were compared with those from early TB (E-TB) patients and healthy tuberculin reactors (HTR). IFN-gamma production was significantly depressed in both CRTB and E-TB groups compared with HTR. In response to the 30-kDa Ag, TNF-alpha levels were significantly depressed only in CRTB patients, while greatly increased in E-TB patients. In addition, IL-10 production was significantly increased in E-TB patients, and PBMC from both E-TB and CRTB patients secreted more IL-6 than HTR. IL-10 neutralization significantly increased TNF-alpha levels, whereas anti-TNF-alpha did not alter IL-10 induction significantly in PBMC from HTR and CRTB patients. Our findings suggest that CRTB patients have depression in both IFN-gamma and TNF-alpha reponses, which might play important roles during chronic M. tuberculosis infection.
Depression* ; Humans ; Immunotherapy ; Interferon-gamma* ; Interleukin-10 ; Interleukin-12 ; Interleukin-6 ; Interleukins ; Mycobacterium tuberculosis ; Tuberculin ; Tuberculosis* ; Tuberculosis, Multidrug-Resistant ; Tumor Necrosis Factor-alpha*

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Anti-candidal Effect of Polyphosphate.

Tae Jin KIM ; Chee Jong JEONG ; Keun Young PARK ; Je Won SHIN ; Jin Yong LEE

Journal of Bacteriology and Virology.2002;32(4):381-392.

Candida albicans is a commensal yeast normally present in small numbers as normal oral flora. In a certain condition, however, the yeast may proliferate and/or become invasive resulting in oral candidiasis such as denture stomatitis, and may even cause life-threatening systemic candidiasis. The present study was undertaken to test whether polyphosphate (polyP), which has been shown to be a strong antibacterial agent against a variety of oral pathogens, has antifungal effect on C. albicans. C. albicans ATCC 90027 was grown in Sabouraud-Dextrose broth with or without polyP. Anti-C. albicans activity of polyPs with various chain lengths was determined by measuring the growth of candidal cells at 540 nm. polyPs with chain length of 3 (polyP3) or higher effectively inhibited the candidal growth when added at the very beginning of the culture, whereas orthophosphate and pyrophosphate failed to do so. At the concentration of 0.05 percent, all the polyPs tested inhibited candidal growth. The effect of polyP65 that showed stronger anti-candidal effect than others at the concentrations tested and of Calgon (hexametapolyphosphate, practical grade) was further examined. The concentration of 0.03 percent was enough for polyP65 and Calgon to suppress candidal growth throughout the 48-h incubation. PolyP65 added to the growing C. albicans at its exponential phase was as much effective in inhibiting the candidal growth as added at the very beginning of the culture. It was found that 93.8 and 96.9 percent of the yeast cells lost their viability when polyP65 was added to growing C. albicans at the concentrations of 0.03 and 0.05 percent, respectively. Intracellular nucleotide release from the candidal cells incubated with polyP65 was only slightly increased and the nucleotide release was not reversed by the addition of divalent metal ions like Mg++ and Ca++. Under the transmission electron microscope, although the majority of growing C. albicans cells appeared to be atypical in their shape in the presence of polyP65, only a small number of the cells were observed to be lysed. The overall results suggest that polyP has a strong fungicidal activity against C. albicans, in which chelation-mediated cell lysis may not play the major role, but other novel mechanisms that possibly affect the viability of the yeast may be involved. Since polyP also has a strong antibacterial effect on oral pathogens, it may well be used for the prevention and treatment of a variety of oral diseases caused by a wide spectrum of microorganisms including C. albicans.
Candida albicans ; Candidiasis ; Candidiasis, Oral ; Ions ; Phosphates ; Polyps ; Stomatitis, Denture ; Yeasts

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Evaluation of rep-PCR Genomic Fingerprinting for the Molecular Systematics Study of Yersinia Species.

Young Chan LIM ; Ki Jeong KIM ; Mi Ok SONG ; Chul Min PARK ; Jung Ae LIM ; Won Young KIM ; Sang In CHUNG

Journal of Bacteriology and Virology.2002;32(4):367-380.

No abstract available.
Classification* ; Dermatoglyphics* ; Yersinia*

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Effect of Salinity, Temperature, and Glucose on the Production of Vibrio vulnificus Hemolysin.

Hyun Soo KIM ; Sung Heui SHIN ; Hae Ryoung PARK ; Shee Eun LEE ; Choon Mee KIM ; Soo Young KIM ; Young Ran KIM ; Hyun Chul LEE ; Sun Sik CHUNG ; Joon Haeng RHEE

Journal of Bacteriology and Virology.2002;32(4):355-366.

Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and 37 degrees C than in 2.5% and 25 degrees C. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and 25degrees C) was changed into that reflecting human body (0.9% salinity and 37 degrees C), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at 37 degrees C. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose- dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.
Ecosystem ; Exotoxins ; Glucose* ; Human Body ; Hydrogen-Ion Concentration ; Metabolism ; Salinity* ; Vibrio vulnificus* ; Vibrio* ; Virulence

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Epidemiologic analysis of Shigella sonnei Isolates by using Antimicrobial Resistance Gene Probes.

Sung Yong SEOL ; Young Sook JEONG ; Hee Young KANG ; Hak Sun YU ; Yoo Chul LEE ; Dong Taek CHO

Journal of Bacteriology and Virology.2002;32(4):347-354.

Thirty-four Shigella sonnei isolates from 6 outbreaks and sporadic cases from May 1999 until January 2000 in Daegu and 9 regions of Gyeongsangbuk-Do were epidemiologically analyzed by plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization with 2 antimicrobial resistance gene probes, tetA and dfrA1. In outbreak cases, resistance pattern in all of the strains was identical: they were resistant to tetracycline (Tc), streptomycin (Sm), sulfisomidine (Su), trimethoprim (Tp), and nalidixic acid (Na). In sporadic cases, Tc, Sm, Su, Na, ampicillin (Ap), and kanamycin (Km) pattern and TcSmSuTpApNa pattern were additionally observed. Isolates from the same outbreak showed identical plasmid profile and PFGE pattern. Most of different outbreak strains and sporadic strains showed different plasmid profiles, and identical or different PFGE patterns, while all of the isolates shared common tetA gene on a non-conjugative 18.3 kbp R plasmid carrying resistance to tetracycline, streptomycin, and sulfisomidine, and dfrA1 gene on the chromosome. Non-conjugative R plasmids derived from all of the isolates were confirmed to be identical by the Southern hybridization analysis of restriction endonuclease treated or non-treated plasmid profiles using the tetA probe. The same strains also reacted with dfrA1 probe at the same-sized DNA fragment (60 kbp) on pulsed-field gel electrophoresis of total genomic DNA. Our findings suggested that epidemic strains of Shigella sonnei prevalent in the Daegu and Gyeongsangbuk-Do area during the test period should have originated from an identical or closely related strain source although most of strains did not show the same plasmid profile and PFGE pattern.
Ampicillin ; Daegu ; Disease Outbreaks ; DNA ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field ; Gyeongsangbuk-do ; Kanamycin ; Nalidixic Acid ; Plasmids ; R Factors ; Shigella sonnei* ; Shigella* ; Streptomycin ; Sulfisomidine ; Tetracycline ; Trimethoprim

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