Prediction of Microbial Infection of Cultured Cells Using DNA Microarray Gene-Expression Profiles of Host Responses

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI).

Prediction Model for the Cellular Immortalization and Transformation Potentials of Cell Substrates

The establishment of DNA microarray technology has enabled high-throughput analysis and molecular profiling of various types of cancers. By using the gene expression data from microarray analysis we are able to investigate diagnostic applications at the molecular level. The most important step in the application of microarray technology to cancer diagnostics is the selection of specific markers from gene expression profiles. In order to select markers of immortalization and transformation we used c-myc and H-ras(V12) oncogene-transfected NIH3T3 cells as our model system. We have identified 8751 differentially expressed genes in the immortalization/transformation model by multivariate permutation F-test (95% confidence, FDR <0.01). Using the support vector machine algorithm, we selected 13 discriminative genes which could be used to predict immortalization and transformation with perfect accuracy. We assayed H-ras(V12)-transfected "transformed" cells to validate our immortalization/transformation classification system. The selected molecular markers generated valuable additional information for tumor diagnosis, prognosis and therapy development.

Study on the Development of a Rapid Detection Method for Pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis by Nested Polymerase Chain Reaction

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.

Development of a Rapid Detection Method for Yersinia pestis by Polymerase Chain Reaction

A polymerase chain reaction (PCR) method for detection of the pathogenic Yersinia pestis from other Yersinia spp. was developed. Five Y. pestis strains, ninety-two other Yersinia species and twenty-four Enterobacteriaceae strains were collected in Korea and from other countries. Oligonucleotide primers were designed from pathogenic gene of antiphagocytic protein capsule gene (fra 1) and plasminogen activator gene (pla). The 428 bp DNA fragment was amplified from five Y. pestis which contained the fra I gene. No product was amplified from other Yersinia species and other strains of the Enterobacteriaceae. The 439 bp DNA fragment was amplified from three K pestis which contained the pla gene. No product was amplified from two Y. pestis, other Yersinia species and other strains of the Enterobacteriaceae. These showed that the designed primers were specific for detection of Y. pestis among other Yersinia species and Enterobacteriaceae strains. Amplification was successful whether the template was derived from purified DNA or from aliquots of boiled bacterial suspension. The detection limits were 100 pg of DNA and 100 colony forming units (CFU) for fra I and 100 pg DNA and 10 CFU for pla, respectively. Our results prove that the PCR method using specific primers for Y. pestis is a rapid and convenient procedure for routine clinical detection and identification of Y. pestis.

A Study on the Loss of Heterozygosity of the p53 Gene in Primary Uterine Cervical Carcinomas / 대한암학회지

PURPOSE: Allelic deletion of p53 tumor suppressor gene have been observed frequently in a variety of human tumors. These losses are believed to contribute to the development of human cancers. But the loss of heterozygosity (LOH) data on chromosome 17p are rare and controversial with respect to cervical carcinomas. So, we tried to elucidate the frequency of p53 locus LOH in primary cervical carcinoma and compared the LOH data with clinicopathological parameters. MATERIALS AND METHODS: In order to detect LOH within one of the well-known tumor suppressor gene, p53, three intragenic polymorphisms (exon 1, exon 4, and intron 6) and one microsatellite distal to the p53 gene (D17S5) were examined. Paired DNA samples from 55 primary uterine cervical carcinomas and normal bloods were studied for the chromosomal allelic loss of p53 gene locus by polymerase chain reaction (PCR), the presence of human papilloma virus (HPV), and the presence of p53 gene point mutation by PCR-single conformation polymorphism (SSCP) analysis. And the relationships between allelic losses of this gene and conventional clinicopathological parameters were evaluated. RESULTS: We could increase the heterozygosity of the p53 gene up to 1 (100%). The observed allelic loss rate of the p53 locus in informative cases was 5.5% (3/55) and the observed allelic loss rate of the D17S5 locus in informative cases was 8.7% (4/46) . Only one of the four patients with LOH at the D17S5 locus showed a concomittant allelic loss of the p53 gene. The overall LOH incidence of the chromosomal region comprising 17p13.1 (p53) to 17p13.3 (D13S5) was 10.9% (6/55). All the samples contained at least one of the oncogenic HPV type 16 and/or 18 sequences. No shifted bands were observed in the PCR-SSCP analysis of the p53 gene. The LOH of the p53 gene was not related to other parameters including clinical stage, histological type, and degree of differentiation. CONCLUSION: Concerning with the results above, we conclude that the allelic imbalance of the p53 gene itself is not implicated as a major contributing factor in the malignant transformation or the tumor progression in HPV-positive cervical cancers. Another putative tumor suppressor gene which has more important function than p53 gene in cervical carcinogenesis might exist between these two loci [p53 (17p13.1) and D17S5 (17p13.3)].

Cytochrome P450 2E1 Polymorphisms and Genetic Susceptibility to Cervical Cancer / 대한암학회지

PURPOSE: Interindividual genetic differences in susceptibility to chemical carcinogens are one of the most important host factors in human cancer. The genetically determined differences in metabolism, related to cytochrome P450 (CYP450) genes have been reported to be associated with various cancer susceptibility. The present study was set up to establish the frequency of the polymorphic genotypes of two CYP450 (CYP2E1/PstI and CYP2E1/DraI) isozymes in Korea, to evaluate a possible increased incidence of the genotype associated with higher cervical cancer risks among Korean cervical cancer patients. MATERIALS AND METHODS: In this study, extracted DNAs from 228 cervical cancer patients and 360 normal healthy controls were analysed with the polymerase chain reaction-restriction fragment length polymosphism (PCR-RFLP) method. RESULTS: In the CYP 2E1 genotypes, detected by PstI or RsaI digestion, there were no statistically remarkable differences between the cervical cancer patients and control groups. And when the cervical cancer patients were divided into subgroups with respect to the age, the frequency of CYP 2E1/PstI polymorphisms in the cervical cancer patients under the 40 years old was not significantly higher compared to the controls or the patients above the 40 years old and, c1/c1 genotype was prominent in this type of polymorphism. The frequency of CYP 2E1/DraI polymorphisms in the cervical cancer patients was not significantly higher compared to the controls, and D/D genotype was prominent in this type of polymorphism. In cervical carcinoma, the polymorphic genotypes of CYP 2E1 were not correlated to other parameters including clinical stage, histological tumor type, and degree of differentiation. CONCLUSION: These results suggest that individuals carrying CYP 2E1/PstI (c1/c1) or CYP 2E1/DraI (D/D) alleles are not genetically susceptible to cervical cancer in Korea.

Glutathione S-Transferase Polymorphisms and Genetic Susceptibility to Cervical Cancer / 대한암학회지

PURPOSE: The identification of genetic traits that predispose individuals to environmentally induced cancers is one of the challanges in the assessment of individual cancer risk. The genetically determined differences in metabolism, related to glutathione S-transferases (GSTs) have been reported to be associated with various cancer susceptibility. The present study was set up to establish the frequencies of the polymorphic genotypes of two GST (GST- mu and GST-theta) isozymes in Korea, to evaluate a possible increased incidence of the genotypes associated with higher cervical cancer risks among Korean cervical cancer patients. MATERIALS AND METHODS: In this study, extracted DNAs from cervical cancer patients (228 for GST-mu and 241 for GST-theta genotypes) and normal controls (360 for GST-mu and 353 for GST-theta genotypes) were analysed with the polymerase chain reaction (PCR). RESULTS: The overall genotype distribution of the GST-theta polymorphisms was not statistically different between the patients and control groups. But, in the GST-mu null genotypes, there were remarkable differences between patients and control groups when the cervical cancer patients were devided into subgroups with respect to the age. The frequency of GST-mu null polymorphisms in the cervical cancer patients under the 40 years old was significantly higher compared to the patients above the 40 years old (0.01