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*

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

A nested polymerase chain reaction (PCR) was applied to detect and identify pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. We used photochemical postamplification procedure with 8-methoxypsoralen to control carryover contamination. Using the ail and inv gene, the sensitivity and specificity of DNA amplification by nested PCR was considerably improved. The amplified fragment sizes were 298 bp for the ail gene and 295 bp for the inv gene. Amplification was successful when the template was derived from three sources: purified DNA, aliquots of boiled bacterial suspension and aliquots of lysed bacterial suspension. The detection limits were 10 fg of DNA and 2 * 10 colony forming units (CFU) for Y. enterocolitica and 10 fg DNA and 2 CFU for Y. pseudotuberculosis.
DNA ; Limit of Detection ; Methoxsalen ; Polymerase Chain Reaction* ; Sensitivity and Specificity ; Stem Cells ; Yersinia enterocolitica* ; Yersinia pseudotuberculosis* ; Yersinia*

Hantaviruses, members of the family Bunyaviridae, are causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaan (HTN) virus, the etiologic agent of clinically severe HFRS in Far East Asia, Russia and the Balkans, was first isolated in 1976 from lung tissues of striped-field mice (Apodemus agrarius) captured in Songnae-ri, Kyungki-do, Korea. Mitochondrial DNA (mtDNA) in vertebrates evolve very rapidly, and hence it can provide a high resolution for distinguishing between closely related organism and species. To clarify the phylogenetic relationship of HTN viruses with their natural rodent host, a part of M segment of HTN virus and 424-nucleotide region of cytochrome b gene of mtDNA were amplified and sequenced from tissues of striped-field mice by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. A 324-nucleotide region of G2 glycoprotein-encoding M segment of HTN virus was amplified from lung tissues of A. agrarius mice, revealed 84-86% sequence similarity with Apodemus-borne HTN virus strains from China. The co-speciation of Apodemus-borne hantaviruses with its natural reservoir rodents, A. agrarius and A. flavicollis, be found. A. agrarius rodent population from South Korea had almost same genetic background irrespective of their geographic origin. HTN virus strains from South Korea shared a common ancestry and were evolutionarily distinct from HTN viruses kom China. We have found no evidence for the presence of phylogenetic relationship of A. agrarius-borne Korean HTN virus strains with the genetic diversity of their rodent host captured in Korea based on cytochrome b gene of mtDNA.
Animals ; Asia ; Balkan Peninsula ; Bunyaviridae ; China ; Cytochromes b ; DNA, Mitochondrial* ; Far East ; Genetic Variation ; Gyeonggi-do ; Hantaan virus* ; Hantavirus ; Hantavirus Pulmonary Syndrome ; Hemorrhagic Fever with Renal Syndrome ; Humans ; Korea ; Lung ; Mice* ; Murinae* ; Polymerase Chain Reaction ; Rodentia* ; Russia ; Vertebrates

Identification of virus is very importnant in aspects of preventive surveillance system of viral infection. The first isolation of echovirus serotype 25 from hand, foot and mouth disease was accomplished in Pusan, 1998. The authors determined some properties of this virus. Two cases of outbreak were confirmed from the stools of a 3 year-old girl and one 4 year-old boy suffering from hand, foot and mouth disease. Both occurred in April. The isolated viruses showed strong cytopathic effect on RD cells, also on HEp-2, and Vero cell lines after 3 days at 34'C, CO incubation. Isolated virus was identified as echovirus serotype 25 by neutralizing antibody test. Electron micrograph of negative-stained echovirus serotype 25 showed non-enveloped, isometric particle and about 30 nm in diameter.
Animals ; Antibodies, Neutralizing ; Busan* ; Child, Preschool ; Enterovirus B, Human* ; Female ; Foot* ; Foot-and-Mouth Disease* ; Hand* ; Hand, Foot and Mouth Disease ; Humans ; Male ; Vero Cells

Vibrio mimicus, marine bacteria pathogenic for fish, can causes acute gastroenteritis in human. Iron limmited condition like in human body, may change the surface structure of V. mimicus. In this study we obse'rved the effect of iron limmited condition on outer membrane protein of V. mimicus. Ethylenediamine-di (O-hydroxy-phenylacetic) acid (EDDA), an iron chelator, delayed the time to reach expotential growth of V. mimicus in brain heart infusion medium from 3 hours to 20 hours. Outer membrane protein of V. mimicus-CON (cultured in BHI) and V. mimicus-EDDA (cultured in BHI contain EDDA) were seperated by 1% sarcosine from total cell envelop. SDS-PAGE of V. mimicus-EDDA and V. mimicus-CON showed similar protein profiles contain 37 kDa major protein but 86 and 90 kDa protein were induced differently. Immunological properties of above protein were determined by ELISA and western blotting. 86 kDa EDDA- specific OMP was induced in V. mimicus (isolate 96-1), V. parahaemolyticus (serotype 09), V. alginolyticus (isolate 95-1), E. coli (human isolate) and V. vulnificus ATCC 27562 in iron limmited condition.
Bacteria ; Blotting, Western ; Brain ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gastroenteritis ; Heart ; Human Body ; Humans ; Iron* ; Membrane Proteins ; Membranes* ; Sarcosine ; Vibrio mimicus* ; Vibrio*

Rapid identification of Mycobacterium spp. isolated from patients is important with increased isolation of mycobacteria other than tubercle bacilli (MOTT). DNA-DNA hybridization with streptavidin-peroxidase and tetramethylbenzidine (TMB) color reaction method was recognized as a useful tool for identification of various species of mycobacteria. In this study, optimum condition of the test was determined. The optimal concentrations of tetramethylbenzidine dihydrochloride and hydrogen peroxide for streptavidin-horseradish peroxidase were 0.3-0.6 ug/ ml and 0.16 mM, respectively. The TMB stock solution was stable when prepared in methanol and the dilution of TBM stock solution in 10 mM sodium citrate-10 mM EDTA solution (pH 5.0) gave highest peroxidase-TMB activity. The suitable composition of hybridization solution consisted of 2 x SSC, 10% dextran sulfate, 50 ug/ml salmon DNA, 5 x Denhardt's solution, and 50% formamide. The 5-minute heating at 100C of test DNA prior to photobiotin labeling significantly increased the reaction. In conclusion, DNA-DNA hybridization method with streptavidin-peroxidase and TMB color reaction method may be useful for rapid identification of Mycobacterium spp. isolated from patients.
Dextran Sulfate ; DNA ; Edetic Acid ; Heating ; Hot Temperature ; Humans ; Hydrogen Peroxide ; Methanol ; Mycobacterium* ; Peroxidase ; Salmon ; Sodium

A total of 100 Vibrio spp. strains were examined for production of various extracellular enzyme and for plasmid content plasmid were subjected to digestion with restriction enzymes. Most of them produced extracellular enzyme more than one, especially V. parahaemolyticus and V. cholerae non-01 strains were showed production of various extracellular enzymes. About the 55% Vibrio spp. have the plasmid more than one, but a lot of Vibrio spp. (about 45%) did not possess any plasmid. Most of these plasmid various derivatives ranged from 2.4 kb-23 kb, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between 70 kb-100 kb). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH, CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 6 strains of environmental isolates. Nine strains of 11 strains, involved in 4 strains of V. parahaemolyticus and 1 strain of V. cholerae non-01 from clinical isolates and 4 strains of V. parahaemolyticus from environmental isolates, could be successfully amplified in 400 bp by PCR, no amplification products were obtained from TDH-negative strains. The PCR results were consistent with DNA hybridization. In the experiments of ctx gene detection, in all, 3 strains of V. cholerae non-01 from clinical isolate and 1 strains of V. cholerae non-01 from environmental isolate were observed CT- positive. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.
Cholera ; Digestion ; DNA ; Plasmids ; Polymerase Chain Reaction ; Vibrio* ; Virulence Factors* ; Virulence*

The gene ftsH encodes a membrane-bound and ATP-dependent protease that is involved in a variety of cellular functions including heat-shock and stress response. Streptococcus pneumoniae DNA encompassing most part of the ftsH gene was cloned in Escherichia coli and sequenced. Due to the unsuccessful cloning as seen in other pneumococcal promoters, the 5'-end of the gene including the upstream promoter region was amplified by inverse polymerase chain reaction and then sequenced by cyclic sequencing. The amino acid sequence that is deduced from the 1,959 bp-long ftsH gene is very similar to FtsH of several gram-positive bacteria and E. coli within the region responsible for the AAA (ATPase associated with diverse cellular activities) function. Except for the N-terminal domain that contains a short extracellular region between two mernbrane-spanning segments, pneumococcal FtsH shows striking sequence similarity to that of a closely related species Lactococcus lactis within the conserved cytoplasmic domain where two ATP-binding motifs, the AAA Signature motif, and a zinc-binding motif are found.
Amino Acid Sequence ; ATP-Dependent Proteases ; Base Sequence* ; Clone Cells* ; Cloning, Organism* ; Cytoplasm ; DNA ; Escherichia coli ; Gram-Positive Bacteria ; Lactococcus lactis ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Streptococcus pneumoniae* ; Streptococcus* ; Strikes, Employee

No abstract available.
Gingival Crevicular Fluid* ; Periodontal Diseases*