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Actinobacillus actinomycetemcomitans, a gram-negative, capnophiTic bacterium, is associated with several human diseases including periodontal disease. Products of A. actinomycetemcomitans exert immunomodulatory effects on various lymphoid populations, some of which may be implicated in the pathogenesis of periodontitis. It has been recently suggested that some of periodontopathic bacterial products might possess superantigenic (SAg) activities. In order to examine SAg activity of A. actinomycetemcomitans, we tried to purify immunomodulating factor (IMF) which can induce proliferation of mouse splenocytes and human PBMC. IMF fraction was obtained from the culture supernatant of A. actinomycetemcomitans by alcohol precipitation, ultrafiltration, size exclusion chromatography, and dye ligand affinity chromatography which has been widely used for the puri5cation of known SAgs. SDS-PAGE analysis showed that the factor migrated to a molecular mass of 40 kDa. The concentration of IMF which elicited maximal proliferative response of mouse splenocytes was ranged 1-10 ug/ml of protein on day 3 in culture. Human PBMC gave a similar response profile to IMF, but their maximal response was obtained by lower concentraion of IMF on day 2 in culture. This activity of IMF was heat and proteinase K sensitive and was not blocked by co-incubation with polymyxin B, a ligand for the lipid A region of lipopolysaccharide. T cell-enriched fraction of mouse splenocytes obtained by nylon wool column lost the response to IMF. Even though mitomycin C-treated antigen presenting cells were added to T cell-enriched fraction, the response to IMF was feeble as compared to unfractionated cells. Splenocytes depleted of T cells by anti-Thy 1.2 and complement also did not respond to IMF. These findings demonstrated that T cells are responsible for a minor proportion of the observed proliferation induced by IMF and the help of these cells are essential to the most of the proliferating cells which may be B cells. This observation was confirmed by flow cytometric analysis of responding lymphocyte subpopulations. These results indicate that IMF of A. actinomycetemcomitans does not act in a manner consistent with known SAgs but is more relevant to the explanation of pathologic findings of periodontal lesions.
Actinobacillus* ; Aggregatibacter actinomycetemcomitans* ; Animals ; Antigen-Presenting Cells ; B-Lymphocytes ; Chromatography, Affinity ; Chromatography, Gel ; Complement System Proteins ; Electrophoresis, Polyacrylamide Gel ; Endopeptidase K ; Hot Temperature ; Humans ; Interleukin-2* ; Lipid A ; Lymphocyte Subsets ; Lymphocytes* ; Mice ; Mitomycin ; Nylons ; Periodontal Diseases ; Periodontitis ; Polymyxin B ; T-Lymphocytes ; Ultrafiltration ; Wool

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Actinobacillus actinomycetemcomitans Indeces Apoptosis of Jurkat Cell Line Through the Cleavage of Poly (ADP-ribose) Polymerase.

Sang Hwa LEE ; Su Yeong SEO ; Su Jin JEONG ; Seung Ho YOO ; Sun Mee PARK ; Min Ho JEONG ; Sung Tae YEE ; Jung Man KIM

Journal of the Korean Society for Microbiology.1998;33(5):507-519.

No abstract available.
Actinobacillus* ; Aggregatibacter actinomycetemcomitans* ; Apoptosis* ; Humans ; Jurkat Cells*

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Epidemiology of Serratia marcescens Isolates by Transferable Resistance Gene Analysis.

Sung Yong SEOL ; Dong Taek CHO ; Yoo Chul LEE ; Haeng Seop SHIN ; Hee Kyung CHANG ; Kyung Sook KIM

Journal of the Korean Society for Microbiology.1998;33(5):485-497.

Conjugative R plasmids derived from 74 clinical isolates of Serratia marcescens were epidemiologically analyzed for antimicrobial resistance, EcoRI restriction endonuclease analysis and Southern hybridization with DHFR, TEM and SHV probe. 1. Resistance frequency of isolates against various B-lactam antibiotics was changed by year. 2. Twenty (27%) resistant strains transferred 32 R plasmids to E. coli or Klebsiella by mixed culture. Most strains isolated from 1994 to 1996 transferred only trimethoprim resistance but most strains isolated from 1997 did resistances against gentamicin (Gm) and B-lactams including ampicillin (Ap), carbenicillin (Cb), cefazolin (Cz), cefaloridine (Cl), cefamandole (Cn). 3. Ten plasmids of GmApCbCzC1Cn or GmApCbCzC1 pattern and 3 plasmids of TcSuGmTbApCbCzC1 pattern respectively showed identical EcoRI restriction endonuclease digestion patterns and hybridized fragment patterns with TEM-1 probe by Southern hybridization. These results indicate that the epidemic plasmids carrying blamM gene were present in this hospital in 1997 and molecular genetic analysis of R plasmids can be used to discriminate S. marcescens isolates for epidemiologic studies.
Ampicillin ; Anti-Bacterial Agents ; Carbenicillin ; Cefamandole ; Cefazolin ; Cephaloridine ; Digestion ; DNA Restriction Enzymes ; Epidemiologic Studies ; Epidemiology* ; Gentamicins ; Klebsiella ; Molecular Biology ; Plasmids ; R Factors ; Serratia marcescens* ; Serratia* ; Trimethoprim Resistance

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IS6110 based DNA Fingerprinting of Mycobacterium tuberculosis Isolated from The Nationwide Tuberculosis Prevalence Survey in 1995.

Sang Jae KIM ; Young Kil PARK ; Sang Hyun CHO ; Gil Han BAI ; Yeun KIM

Journal of the Korean Society for Microbiology.1998;33(5):475-484.

One hundred and thirty-eight strains of Mycobacterium tuberculosis isolated from the 7th nationwide tuberculosis prevalence survey in 1995 were subjected to the restriction fragment length polymorphism (RFLP) analysis using IS6110 probe to define the representative fingerprinting patterns of Korean strains of M. tuberculosis and to evaluate the usefulness of DNA fingerprinting in tracing the transmission link of M. tuberculosis. Among 138 strains, 129 different IS6l10 RFLP types were identified. The number of bands in IS6110 RFLP types diversed from 1 to 20, and the majority (75%) was 9 to 14 bands. The RFLP patterns of 8 out of 15 strains isolated from the follow-up survey of one and half year later after the 7th national TB prevalence survey were unchanged when compared with previous RFLP patterns. Fifteen (11%) out of 138 strains were grouped in 6 IS6110 clusters; 2 with 10 copies, 2 with 12 copies, 1 with 14 copies, and 1 with 17 copies. These clusters were unable to be subclassified by IS1081 or (GTG) probes except one cluster by pTBN12 probe. The transmission links of 2 clusters were deducible; one from household and another from neighborhood, but those of remaining clusters were unclear because they had no contact one another. The results suggest that vigorous transmissions in tuberculosis are still progressing in Korea.
Dermatoglyphics ; DNA Fingerprinting* ; DNA* ; Family Characteristics ; Follow-Up Studies ; Korea ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Restriction Fragment Length ; Prevalence* ; Residence Characteristics ; Tuberculosis*

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Cloning and Sequencing Analysis of the Gene Encoding the 66-kDa Protein in Borrelia hermsii.

Yoon Hoh KOOK ; Kyung Hee PARK ; Seung Hyun LEE ; Jong Hyun KIM

Journal of the Korean Society for Microbiology.1998;33(5):463-473.

The gene encoding the 66 kilodalton (kDa) protein of Borrelia hermsii HS1 was cloned and sequenced. Chromosomal DNA was prepared from purified B. hermsii and used in construction of genomic library. The library was screened for positive clones by 314 bp DIG-labeled probe synthesized on the basis of the part of the sequence of B. hermsii. Positive clone was subcloned into p2ErO vector and was designated as pBH11. pBH11 were subcloned into pBluscript vector and were designated as pBH11-1 (500 bp), pBH11-2 (800 bp), pBH11-3 (600 bp) and pBH11-4 (800 bp). The plasmids were sequenced and determined the nucleotide sequence of p66. The open reading frame of the p66 consisted of 1803 base pairs coding for 600 amino acid protein. The basic information on the p66 gene of B. hermsii HS1 obtained from this study will be useful for further analysis and experiment of pathogenesis of the borrelia.
Base Pairing ; Base Sequence ; Borrelia* ; Clinical Coding ; Clone Cells* ; Cloning, Organism* ; DNA ; Genomic Library ; Open Reading Frames ; Plasmids

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The Multiplex RT-PCR for the Serotyping of Hantaviruses.

Pyung Woo LEE ; Man Seong PARK

Journal of the Korean Society for Microbiology.1998;33(5):451-461.

Hantaviruses are negative-strand RNA viruses that contain three segmented genome and belong to the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, these viruses have a possibility to produce reassortant that have genomic sets mixed with different segments originated from both parental virus during the genetic interaction. To investigate whether this phenomenon occurs actually, Hantaan virus (HTN) could be coinfected with Seoul virus (SEO) to Vero-E6 cell. And also, HTN could be coinfected with Prospect hill virus (PH) to investigate the rate of genetic reassortment according to the genetic distance among the HTN, SEO and PH viruses. As a available method to differentiate the reassortant, we used the multiplex RT-PCR that detect and differentiate the serotype of hantaviruses in mixed infection. Each progeny clone could be screened by multiplex RT-PCR. So, we have constructed the multiplex RT-PCR system that separated amplifications for each segment of Hantaviruses. Our multiplex RT-PCR system provide sensitive, specific and simplified tools for the rapid differentiation of hantaviruses serotypes and also the diagnosis of Hantavirus infections.
Bunyaviridae ; Clone Cells ; Coinfection ; Diagnosis ; Genome ; Hantaan virus ; Hantavirus Infections ; Hantavirus* ; Humans ; Hydrogen-Ion Concentration ; Parents ; RNA ; RNA Viruses ; Seoul virus ; Serotyping*

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Cloning and Sequence Analysis of Gene Encoding an Outer Membrane Protein ( OmpTL ) of Treponema Strain PFB4G Isolated from Periodontitis Patients.

Bong Kyu CHOI ; Kwang Kyun PARK

Journal of the Korean Society for Microbiology.1999;34(2):201-209.

Treponema strain PFB4G is a novel oral spirochete and one of the most frequently detected organisms in subgingival plaque samples from rapidly progressive periodontitis and adult periodontitis patients. In this study, a genomic library of Treponema strain PFB4G was constructed in lambdaZAP expression vector. One positive clone that carried a 2.6-kb fragment was identified by screening with chicken Ig Y (immunoglobulin yolk) antibody raised against whole bacterial sonicates. Nucleotide sequencing of the subclones revealed an open reading frame (ORF) lacking the 5'-end. This region was obtained by PCR amplification using a degenerative and a specific primer. A complete open reading frame of 1,770 bp was identified and the deduced polypeptide consisted of 590 amino acids with a molecular mass of 65 kDa. The polypeptide, designated as OmpTL, had a typical prokaryotic signal sequence (19 amino acids) with a potential cleavage site for signal peptidase I and showed a significant level of homology with the outer membrane proteins of other oral treponemes, especially with that of Treponema maltophilum. The isolation of the gene encoding an outer membrane protein may allow the study of their roles in future, possibly as adhesion, pore forming or induction of proinflammatory cytokine synthesis.
Amino Acids ; Chickens ; Chronic Periodontitis ; Clone Cells* ; Cloning, Organism* ; Genomic Library ; Humans ; Mass Screening ; Membrane Proteins* ; Membranes* ; Open Reading Frames ; Periodontitis* ; Polymerase Chain Reaction ; Protein Sorting Signals ; Sequence Analysis* ; Spirochaetales ; Treponema*

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Genotyping and Molecular Epidemiology of Helicobacter pylori Strains Isolated from Korea.

Kwang Ho RHEE ; Woo Kon LEE ; Myung Je CHO ; Seung Chul BAIK ; Hee Shang YOUN ; Young Seok JEON ; Yeo Jeong CHOI ; Jae Young SONG ; In Girl LEE ; Sang Haeng CHOI ; Byung Sang LEE

Journal of the Korean Society for Microbiology.1999;34(2):189-199.

This study aims to know cagA and vacA genotypes and the molecular epidemiology of H. pylori strains isolated from patients with gastroduodenal disorders and normal healthy persons of Korea using PCR genotyping, RAPD fingerprinting, and PCR-RFLP. PCR genotyping for cagA genotyping showed that 143 H. pylori isolates tested in this study were cagA positive strains. All the isolates were confirmed as type sla genotype and 13 isolates (9%) among of 143 strains were confirmed as containing the RS2 element. All 143 isolates showed individually unique RAPD profiles. PCR-based RFLP was done to assess the sequence diversity of H. pylori flagella genes. From all H. pylori isolates, 30 distinct patterns were found with HhaI digestion of 1.5 kb flaA segment and 12 distinct patterns were produced with MboI digestion. Among 30 persons, from whom multiple isolates could be obtained, 27 (90%) were confirmed to be colonized with an identical H. pylori strain and 3 (10%) were shown to be infected with the different strains. Among 5 persons attended in follow-up study, 4 were infected with identical strain for 1 year, 1 carried different strains after 1 year. Genotypes of isolates recovered from children were shown to be identical to those of their parents, suggesting that children acquire H. pylori infection from their parents.
Child ; Colon ; Dermatoglyphics ; Digestion ; Flagella ; Follow-Up Studies ; Genotype ; Helicobacter pylori* ; Helicobacter* ; Humans ; Korea* ; Molecular Epidemiology* ; Parents ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

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