We purified enolase from Candida albicans KNIH10 strain which was isolated from a clinical specimen in Korea. The purified enolase was used to detect anti-Candida antibodies in sera of patients with invasive candidiasis. For purification of enolase from the crude extract prepared by French pressure at 20,000 PSI, the fast performance liquid chromatography (FPLC) using DEAE-sepharose column was used. The elutes at 0.3-0.4 M NaCl in FPLC was purified with homogenity in SDS-PAGE and its enzymatic activity was confirmed in sera of invasive candidiasis with candidemia patient by immunoblotting. The purified enolase indicated no siggnal (100% specificity) in 40 normal human sera and 75% (6/8) sensitivity in sera of candidemic patients with suspicious invasive candidiasis by immunoblotting.
Antibodies ; Candida albicans* ; Candida* ; Candidemia ; Candidiasis, Invasive ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Humans ; Immunoblotting ; Immunologic Tests* ; Korea* ; Phosphopyruvate Hydratase*

Acinetobacter baumannii strains are emerging pathogens of the nosocomial infection with an increasing frequency in recent years. The therapeutic difficulty due to the wide spread of multiple resistant strains was major problem in A. baumannii infection. It seems likely that high frequency of A. baumannii infection will be increasing epidemiological importance in the future. However, the current limited understanding of the epidemiology of A. baumannii infections is caused by lack of a rapid and practical method for the molecular characterization of A. baumannii strains. This study was undertaken to determine molecular types and genetic similarity among A. baumannii strains isolated from four hospitals by RAPD analysis. Eighty-five strains, including 40 from Chunnam University Hospital, 27 from Dankook University Hospital, 15 from Yonsei University Hospital, and 3 from Seonam University Hospital, were classified into three molecular types. Molecular type II was the most common pattern and included 72 strains. All strains from Dankook University Hospital and 40 strains from Chunnam University Hospital belonged to molecular type I or II. A. baumannii strains form Yonsei University Hospital were very distant similarity values. The range of genetic similarity values among 85 strains of A. baumannii was 0.26 to 1.00. Although phenotypes including biotype and antimicrobial resistance pattern of A. baumannii strains were same or very similar to each other, their RAPD patterns were quite different. Typing with phenotypes was found to be less reliable than molecular typing by RAPD analysis. These results suggest that RAPD analysis provides rapid and simple typing method of A. baumannii strains for epidemiological studies. This work is the first epidemiological report of A. baumannii infections in Korea and it is hoped that results of this work may contribute to a better understanding of the clinical importance and epidemiology of A. baumannii strains.
Acinetobacter baumannii* ; Acinetobacter* ; Cross Infection ; DNA* ; Epidemiology ; Hope ; Korea ; Molecular Typing* ; Phenotype

The non-ELR-containing CXC chemokines Mig and IP-10 have been shown to function as chemotactic cytokines for activated T lymphocytes. In this study, we examined the potential involvement of Mig and IP-10 in antimycobacterial response of mice immunized or infected with M. bovis BCG. The accumulation of Mig and IP-10 mRNA in resident peritoneal monocytes (RPMPHI) was slightly reduced by stimulation with vBCG, and the degree was greater for 24 hr culture even though IFN-gamma was added. Expression of Mig, IP-10, and IFN-gamma in 24 hr delayed-type hypersensitivity (DTH) response was stronger in vBCG-immune mice than in the non-immune. The increase of DTH measured by foot-pad thickness appears to be clearly related to the levels of chemokines Mig and IP10 messages and those of IFN-gamma and IL-12. Stimulation with vBCG for 2 days decreased or completely dropped the levels of Mig message in non-immune or immune splenocytes, respectively, whereas IP-10 message was slightly decreased in 2 days culture. Moreover, messages for IL-12 (p40) showed similar kinetics for Mig. The levels of Mig and IP-10 mRNA during the course of infection with BCG were not readily changed in lungs, livers, and spleens from BCG-infected mice. Although there was no obvious changes of Mig and IP-10 messages in the target organs during infection process, we found that the infection progressed over the first 3 wk before being contained by the emerging immune response suggested from detectable amount of IFN-gamma mRNA around this time. In view of selectivity of chemokines Mig and IP-10 for activated T cells, these data suggest that chemokine Mig and IP-10, especially in collaboration with IL-12 and IFN-gamma, may play a role as T cell recruiters in immune response against mycobacterial infection.
Animals ; Chemokines ; Chemokines, CXC* ; Cooperative Behavior ; Hypersensitivity ; Interleukin-12 ; Kinetics ; Liver ; Lung ; Mice* ; Monocytes ; Mycobacterium bovis* ; RNA, Messenger ; Spleen ; T-Lymphocytes

FtsH is a membrane-bound, ATP-dependent metalloprotease that is involved in a variety of cellular functions including the regulation of responses to heat and stress shock. Previously, we had cloned and sequenced pneumococcal ftsH gene whose deduced amino acid sequence was very similar to those of several gram-positive bacteria and Escherichia coli, except for the N-terminal domain that was responsible for membrane anchoring. In order to better understand the role of Streptococcus pneumoniae FtsH, we expressed pneumococcal ftsH gene in Escherichia coli. When it was expressed from a strong promoter, Ptac, a considerable amount of the recombinant FtsH was produced, although the prolonged induction resulted in not only accumulation of breakdown products but also ceasing of the further growth of E. coli host. This indicated that the expression of the exogenous ftsH gene was tightly regulated since the excessive FtsH appeared detrimental to bacterial cells. In Western blotting, the pneumococcal FtsH protein, whether native or recombinant, was reactive to anti-E. coli FtsH serum. The observation that FtsH proteins were well conserved throughout the bacterial kingdom and its expression level was fine-tuned suggests an important role for this protein in the stress adaptation which may be related to infecting process by pneumococci.
Amino Acid Sequence ; Blotting, Western ; Clone Cells ; Escherichia coli ; Gram-Positive Bacteria ; Hot Temperature ; Membranes ; Shock ; Streptococcus pneumoniae* ; Streptococcus*

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer (9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)). The extract (10 mug) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.
Electrophoresis ; Electrophoresis, Gel, Two-Dimensional* ; Helicobacter pylori* ; Helicobacter* ; Hydrogen-Ion Concentration ; Molecular Weight ; Protein Isoforms ; Proteomics ; Proton-Motive Force ; Silver Staining ; Sodium ; Urea

In this study, the distribution of the mec regulator genes and the presence of the mutation in mecI gene and mec promoter region among 50 MRSA clinical isolates derived from a single university hospital in Korea were analyzed. Among 50 MRSA strains, 13 strains had a deletion of mecI gene, and 37 strains were found to have mutations in mecI gene or mecA promoter region corresponding to a presumptive operator of mecA, i.e., the binding site of the repressor protein. Furthermore, in order to track the evolution of methicillin-resistant Staphylococcus aureus (MRSA) distributed in Korea, we determined the MRSA clonotype by combined use of genetic organization patterns of mec regulator genes, ribotype, and coagulase type. As the result, 48 of 50 MRSA strains could be classified into four distinct clones. Clonotype I is characterized by the coagulase type 3, deletion of mecI gene, and ribotype 1 shared by NCTC10442, the first reported MRSA isolate in England (9 strains). Clonotype II is characterized by the coagulase type 4, C to T substitution at position 202 of mecI gene, and ribotypes 2, 3 and 4 shared by 85/3619 strain isolated in Austria (10 strains). Clonotype III is characterized by the coagulase type 2, mutations of mecA promoter region and/or mecI, and ribotypes 4, 5, and 6 shared by N315 strain isolated in Japan (25 strains). Clonotype IV is characterized by the coagulase type 4, deletion of mecI gene, and ribotype 7 (4 strains). The clonality of two strains could not be determined due to their undefined ribotype.
Austria ; Binding Sites ; Clone Cells ; Coagulase ; England ; Genes, Regulator ; Japan ; Korea* ; Methicillin Resistance* ; Methicillin-Resistant Staphylococcus aureus* ; Promoter Regions, Genetic ; Ribotyping

Porphyromonas gingivalis has been implicated in periodontal diseases. Accumulating evidence suggests that cardiovascular disease is the most prevalent medical problem in patients with periodontal diseases. In order to check the possibility that P. gingivalis is involved in coronary heart disease, the present study was performed to observe P. gingivalis adherence and invasion of human coronary artery endothelial cells (HCAEC) and production of cytokines and growth factors by HCAEC upon P. gingivalis infection. 3H-labeled P. gingivalis 381 was incubated with HCAEC for 90 min. The radioactivity of the washed HCAEC was a measure of the absorbed (adhering and invading) P. gingivalis. The absorption radioactivity of the HCAEC infected by P. gingivalis was determined to be 59.58% of the input bacterial cells. In contrast, the absorption radioactivity of the cells infected by S. gordonii Challis which was employed as a control was negligible (0.59%). DPG3, a P. gingivalis mutant defective of fimbriae, appeared to be impaired to some extent in capability of adherence/invasion as compared to that of the parental strain 381, showing 43.04% of the absorption radioactivity. The absorption radioactivity of the HCAEC infected by P. gingivalis 381 in the presence of excessive fimbriae at the concentrations of 50 mug and 100 mug/ml was 57.27 and 45.44%, respectively. Invasion of HCAEC by P. gingivalis 381 was observed by an antibiotic (metronidazole) protection assay and transmission electron microscopy (TEM). In the antibiotic protection assay, invasion by the bacterium was measured to be 0.73, 1.09, and 1.51% of the input bacterial cells after incubation for 30, 60, and 90 min, respectively. Invasion by DPG3 was shown to be 0.16% after 90-min incubation. In comparison of invasion efficiency at 90 min of the incubation, the invasion efficiency of DPG3 was 0.37% while that of its parental strain 381 was 2.54%. The immunoblot analysis revealed fimbriae of P. gingivalis did not interact with the surface of HCAEC. These results suggest that fimbriae are not the major contribution to the adherence of P. gingivalis to HCAEC but may be important in the invasion of HCAEC by the bacterium. The presence of cytochalasin D (1 mug/ml) and staurosporine (1 muM) reduced the invasion of HCAEC by P. gingivalis 381 by 78.86 and 53.76%, respectively, indicating that cytoskeletal rearrangement and protein kinase of HCAEC are essential for the invasion. Infection of P. gingivalis induced HCAEC to increase the production of TNF-alpha by 60.6%. At 90 min of the incubation, the HCAEC infected with P. gingivalis cells was apparently atypical in the shape, showing loss of the nuclear membrane and subcellular organelles. The overall results suggest that P. gingivalis may cause coronary heart disease by adhering to and invading endothelial cells, and subsequently damaging the cells.
Absorption ; Cardiovascular Diseases ; Coronary Disease* ; Coronary Vessels ; Cytochalasin D ; Cytokines ; Endothelial Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Microscopy, Electron, Transmission ; Nuclear Envelope ; Organelles ; Parents ; Periodontal Diseases ; Porphyromonas gingivalis* ; Porphyromonas* ; Protein Kinases ; Radioactivity ; Staurosporine ; Tumor Necrosis Factor-alpha

No Abstract Available.
Helicobacter pylori* ; Helicobacter*

No Abstract Available.
Clone Cells* ; Cloning, Organism* ; Mycobacterium tuberculosis* ; Mycobacterium*

No Abstract Available.
Astrocytes* ; Humans*