Comparison of Real-Time Quantitative PCR with Northern Hybridization for Quantification of CgCDR1 and CgCDR2 Gene Expression in Candida glabrata / 대한의진균학회지

BACKGROUND: One of main mechanisms responsible for acquired azole resistance of Candida glabrata is the increased drug efflux mediated ABS transporters, which are encoded by CgCDR1 and CgCDR2 genes. OBJECTIVES: We compared real-time reverse transcriptase PCR (RT-PCR) with northern hybridization for quantitative analysis of CgCDR1 and CgCDR2 expression in bloodstream isolates of C. glabrata. METHODS: Nineteen blood isolates of C. glabrata were selected, including nine fluconazole susceptible (MIC < or =8 microgram/ml), nine susceptible dose-dependent (S-DD, MIC 16~32 microgram/ml), and one resistant (MIC 128 microgram/ml), isolates. The expression of CgCDR1 and CgCDR2 was quantified using real-time RT-PCR with ROTOR Gene 3000 (Corbettet research, Austria). The results were compared with northern hybridization with sequence-specific probes. RESULTS: Correlation of quantification results between real-time RT-PCR and northern hybridization yielded correlation coefficients of 0.92 for CgCDR1 and 0.82 for CgCDR2 gene. By both methods, no significant differences were observed in the levels of expression of CgCDR1 and CgCDR2 between fluconazole-susceptible isolates and S-DD isolates. In contrast, a strain with high fluconazole resistance (MIC 128 microgram/ml) revealed a greater abundance of CgCDR1 by both methods, compared to the other isolates. Conclusion: This study show that real-time PCR method for C. glabrata RNA quantification correlates well with traditional northern hybridization and can be a valuable alternative to northern hybridization for rapid quantification of CgCDR1 and CgCDR2 genes in clinical isolates of C. glabrata.

Cloning of novel gene GP1 associated with gastric cancer and its premalignant lesions / 基础医学与临床

Objective To explore the differentially expressed genes of gastric cancer,matched normal gastric tissue and premalignant lesions. Methods The differentially expressed cDNA bands were isolated and identified by fluorescent differential display and then reamplified by PCR.After being cloned,all cDNA fragments were sequenced. Through BLAST,the results were compared with GenBank database for homology analysis. The expression of the cDNA fragment in different tissues was confirmed by Northern blot. SMART-RACE(rapid amplication of cDNA ends) was used to amplify the full length cDNA sequence. Bioinformatics analysis was performed to analysis its function. Results A differentially expressed cDNA fragment expressed lower in gastric cancers and premalignant lesions,no homology was found in GenBank. The novel cDNA fragment was given an accession number of EST(CD454989)in GenBank. A full length cDNA sequence of 1362 bp was acquired,who coded 267 amino acids,and was homologous with BAA91562.1,whose physiology function was unknown. Conclusion A differentially expressed gene was found and it may be involved in process of the gastric cancer.

The Relation between the High-level Resistance to Fluoroquinolones and the Over-expression of the acrA among Quinolone-Resistant Escherichia coli-Quantification of acrA by Using Real time PCR and Northern hybridization / 감염과화학요법

BACKGROUND: Target point mutation of DNA topoisomerase, which is the typical mode of quinolone resistance, cannot explain high level resistance to quinolones. Therefore, many authors looked into over expression of efflux pump as the possibility. After quantificating the arcA mRNA, which controls AcrAB- TolC, the authors tried to find out the difference in the expression of arcA mRNA according to MIC of ciprofloxacin. The authors also tried to determine the usefulness of real time PCR, which is more reproducible and takes less time than preexisting immunoblot assay, through quantification of acrA. MATERIAL AND METHODS: Mutations in topoisomerase (GyrA, ParC) of 20 quinolone resistant E. coli isolates were identified by PCR and direct DNA sequencing. AcrA level was measured by real time PCR. GAPDH of E.coli was used as endogenous control. The expression of acrA was confirmed through northern hybridization method, the results obtained by real time PCR were compared. RESULTS: 1) Topoisomerase mutations were found in all quinolone resistant E. coli strains. 2) AcrA expression in fluoroquinolone-resistant E. coli was quantified by using real time PCR. There was no relationship between the ratio of acrA expression to GAPDH and MIC of ciprofloxacin. 3) With Northern hybridization, we compared the band of acrA to that of GAPDH in compactness and area. No difference in the expression according to MIC could be found. 4) The results of AcrA/GAPDH were significantly correlated between the real-time PCR and northern blot (P<0.05, correlation coefficiency 0.98). CONCLUSION: In this study, no relationship between overexpression of AcrA gene and high level fluoroquinolone resistance. Therefore, we assume that mechanism other than AcrAB efflux pump is involved in and contribute to high-level fluoroquinolone resistance. However, the degree of efflux pump expression could be confirmed with real time PCR using acrA mRNA. Therefore, real time PCR could be used in the molecular biologic study on the mechanism of resistance to antibiotics.

The Relation between the High-level Resistance to Fluoroquinolones and the Over-expression of the acrA among Quinolone-Resistant Escherichia coli-Quantification of acrA by Using Real time PCR and Northern hybridization / 감염과화학요법

BACKGROUND: Target point mutation of DNA topoisomerase, which is the typical mode of quinolone resistance, cannot explain high level resistance to quinolones. Therefore, many authors looked into over expression of efflux pump as the possibility. After quantificating the arcA mRNA, which controls AcrAB- TolC, the authors tried to find out the difference in the expression of arcA mRNA according to MIC of ciprofloxacin. The authors also tried to determine the usefulness of real time PCR, which is more reproducible and takes less time than preexisting immunoblot assay, through quantification of acrA. MATERIAL AND METHODS: Mutations in topoisomerase (GyrA, ParC) of 20 quinolone resistant E. coli isolates were identified by PCR and direct DNA sequencing. AcrA level was measured by real time PCR. GAPDH of E.coli was used as endogenous control. The expression of acrA was confirmed through northern hybridization method, the results obtained by real time PCR were compared. RESULTS: 1) Topoisomerase mutations were found in all quinolone resistant E. coli strains. 2) AcrA expression in fluoroquinolone-resistant E. coli was quantified by using real time PCR. There was no relationship between the ratio of acrA expression to GAPDH and MIC of ciprofloxacin. 3) With Northern hybridization, we compared the band of acrA to that of GAPDH in compactness and area. No difference in the expression according to MIC could be found. 4) The results of AcrA/GAPDH were significantly correlated between the real-time PCR and northern blot (P<0.05, correlation coefficiency 0.98). CONCLUSION: In this study, no relationship between overexpression of AcrA gene and high level fluoroquinolone resistance. Therefore, we assume that mechanism other than AcrAB efflux pump is involved in and contribute to high-level fluoroquinolone resistance. However, the degree of efflux pump expression could be confirmed with real time PCR using acrA mRNA. Therefore, real time PCR could be used in the molecular biologic study on the mechanism of resistance to antibiotics.

Lactase mRNA Expression in Small Intestines of Korean Fetuses and Adults / 대한내과학회지

OBJECTIVE: The specific activity of lactase-phlorizin hydrolase (LPH) is very high at birth and sharply declines after weaning, producing lactose intolerance. The prevalence of lactose intolerance is up to 85% in Korean adults. Molecular basis of the regulatory mechanisms responsible for the decline of LPH specific activity is still unknown. In order to elucidate the molecular mechanisms regulating the LPH expression during development, LPH specific activity and mI4NA level of Korean fetal and adult intestines were compared. METHODS: 20 fetal small intestines (16-27 weeks) were obtained during therapeutic abortion and were divided into 3 equal length. 20 adult jejunal tissues were obtained from patients without small intestinal disease during laparotomy. Mucosal homogenates were prepared for dissacharidases specific activities measurement and total RNA was extracted for northern and slot hvbridization. LPH mRNA level was measured by laser densitometer. RESULTS: LPH specific activities of proximal, middle and distal portion of fetal intestines (n=20) were 36.2 +/- 22.5, 38.6 +/- 23.2 and 23.2 +/- 19.9 mu/mg protein, respectively. LPH specific activity of adult jejunum (n=8) was 5.9 +/- 1.8 mu/mg protein and significantly (p<0.05) lower than those of fetal intestines. However, there was no significant difference in sucrase and trehalase specific activities between fetal intestines and adult jejunum. Although LPH specific activity of adult jejunum was lower than those of fetal intestines, LPH mBNA level of adult jejunum was as high as those of fetal intestines. CONCLUSION: These results show that LPH specific activity and mRNA level do not parallel, indicating the posttranscriptional control of fetal development of LPH expression.