PURPOSE: Recently, two alternatively spliced survivin variants, survivin-DeltaEx3 and survivin-2B, were identified in a single copy of the survivin gene. It has been reported that the expressions of survivin splice variants significantly correlates with the clinical results in many types of human carcinoma. We investigated the transcription levels of survivin and its splice variants in human colorectal carcinomas, and analyzed correlations between survivin expression levels and clinicopathologic features. METHODS: We used Western blot and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to analyze the protein and mRNA expression levels of survivin variants in 51 colorectal carcinomas. The quantitative RT-PCR was performed using primer pairs specific for survivin and each of its splice variants, then normalized for the gene that encodes glyceraldehydes-3-phosphate dehydrogenase. RESULTS: In Western blotting, the protein levels of survivin were higher in the tumor tissue than in normal tissue. The expression of survivin, survivin-2B and survivin-DeltaEx3 mRNA was present in 96%, 64.7%, and 82.4% of the samples, respectively. When the pathologic parameters were compared, colorectal cancers of advanced pT stages showed significant decrease in survivin-2B mRNA expression by the quantitative RT-PCR (P < 0.001). CONCLUSION: The decreased expression of survivin-2B might be related to tumor progression in colorectal cancers. This finding indicates that alternatively spliced variants of survivin may be involved in refining the functions of survivin during tumor progression.
Blotting, Western ; Coat Protein Complex I ; Colorectal Neoplasms ; Humans ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger

NORs are loops of DNA which occur in nucleoli and which posses ribosomal DNA genes. Ribosomal DNA genes transcribe to ribosomal RNA by RNA polymerase I and are of vital significance in the ultimate synthesis of protein. Proteins associated with the NORs are stained with silver nitrate (Ag-NORs). Ag-NORs were studied in various tumors and might reflect the activity of cells and might be an indicator of the degree of malignancy in tumors. Specimens of prostate obtained by transurethral resection or fine needle biopsy were examined with regard to the number and area of NORs in order to find out whether the above mentioned two parameters reflect proliferative activity in 23 cases of prostatic adenocarcinoma and 10 cases of benign prostatic hyperplasia as controls. The mean number of Ag-NORs in BPH, well, moderate and poorly differentiated adenocarcinoma was 1.24+/-0.10 (mean+/-SD; n=10), 1.87+/-0.23 (n=6). 2.08 J=0.28 (n=10). 4.02+/-0.22 (n=7). respectively. In each group, significant difference was found statistically (p<0.05) except between well and moderate differentiated adenocarcinoma. The mean area of Ag-NORs calculated by morphometry in BPH, well, moderate and poorly differentiated adenocarcinoma was 5.40+/-1.8 um2, 7.84+/-3.68 um2. 14.02+/-4.95 um2, 23.99+/-6.94 um2, respectively and significant difference was found statistically in each group (p<0.05). We concluded that the number and area of NORs increased with degree of malignancy and may reflect proliferative activity of cells in prostatic adenocarcinoma. Further investigations may be needed to confirm whether the number or area of Ag-NORs may be available as a prognostic factor for patient`s survival.
Adenocarcinoma* ; Biopsy, Fine-Needle ; DNA ; DNA, Ribosomal ; Nucleolus Organizer Region* ; Prostate ; Prostatic Hyperplasia ; RNA Polymerase I ; RNA, Ribosomal ; Silver Nitrate

PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. RESULTS: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at 1 and 3 days after irradiation in the 1 Gy exposed group compared with the control group. CONCLUSION: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.
Alkaline Phosphatase* ; Cell Line* ; Cesium ; Collagen Type I* ; DNA, Complementary ; Osteoblasts* ; Polymerase Chain Reaction ; RNA ; RNA, Messenger*

Nucleolar organizer regions(NORs) are argyrophilic and have a loops of DNA that tran scribes to ribosomal RNA by RNA Polymerase I and are involved in formation of nucleolus. They are used in measuring proliferative activity of cells. Mean AgNORs count is increased or large and NORs are irregular in shape in malignant tumors. To examine the relationship of AgNORs to the survival rate of the patients with invasive colorectal adenocarcinoma, we counted mean AgNORs in 200 tumor cells and mean AgNORs#(mean AgNORs in counting AgNORs of the upper 40 nuclei after sorting AgNORs counts of 200 cell nuclei in descending order) after AgNOR staining using 45 cases of formalin-fixed, paraffin-embedded tissue surgically excised at Kang-Dong Sacred heart hospital. The survival rates were not related to age, sex, locations, differentiation, mAgNORs and mAgNORs# except Dukes'stages. There were only statistical significances in AgNORs# between high and low age group(p=0.0001), and between colon and rectum(p=0.005). But it is known that there could be different results according to staining time, fixation and preservation of tissues, counting methods in measuring AgNORs. There might be clinical usefulness of AgNOR if more cases are examined together with better tissue processing and staining methods.
Adenocarcinoma ; Cell Nucleus ; Colon ; Colorectal Neoplasms ; DNA ; Heart ; Humans ; Nucleolus Organizer Region ; RNA Polymerase I ; RNA, Ribosomal ; Survival Rate*

Scleroderma (SSc) renal crisis has been reported to be associated with anti-RNA polymerase I and III (RNAP I/III) antibodies in Caucasians and the Japanese. However, no report is available for Korean SSc patients. Here, we describe the case of a 65-yr-old female SSc patient who developed renal crisis and whose serum contained anti-RNAP I/III antibodies. She was finally diagnosed as having diffuse cutaneous SSc based on skin thickening proximal to the elbows and knees. Sudden hypertension, oliguria, and pulmonary edema were features of her renal crisis. Despite the use of captopril and adequate blood pressure control, her renal function deteriorated. Subsequent renal biopsy findings showed severe fibrinoid necrosis with luminal obliteration in interlobar arteries and arterioles consistent with SSc renal crisis. Serum anti-RNAP I/III antibodies were detected by radioimmunoprecipitation. This is the first report of a renal crisis in a Korean SSc patient with RNAP I/III antibodies.
Scleroderma, Systemic/*complications/enzymology/*immunology ; RNA Polymerase III/*immunology ; RNA Polymerase I/*immunology ; Korea ; Kidney Failure, Acute/*complications/enzymology/*immunology ; Humans ; Female ; Critical Care ; Autoantibodies/*blood ; Aged

Nucleolar organizer regions (NOR) are loops of ribosomal DNA (rDNA) which are transcribed by RNA polymerase I. They produce ultimately ribosome and protein. Thus they are believed to reflect nuclear activity. We applied silver colloid staining technique to human glioma to examine relationship between the mean number of Ag-NOR and histopathological grading. The mean number of Ag-NOR (±S. E of the mean) were 1.17±0.07 in normal brain, 1.53±0.25 in astrocytoma, 2.37±0.71 in malignant astrocytoma. And 2.88±0.41 in glioblastoma multiforme. And there was a statistically significant difference among these. The results show that Ag-NOR technique is a rather simple and rapid method and will become a helpful tool for estimation of the proliferative potential of glioma.
Astrocytoma ; Brain ; Colloids ; DNA, Ribosomal ; Glioblastoma ; Glioma* ; Humans ; Methods ; Nucleolus Organizer Region* ; Ribosomes ; RNA Polymerase I ; Silver

BACKGROUND AND OBJECTIVES: Vasospastic angina (VA) is a specific type of coronary artery disease and develops as a result of coronary artery spasm. Recently, a few studies have revealed that VA caused by coronary artery spasm is related to genetic traits. The objective of this study was to use the recently developed technique of array comparative genomic hybridization (CGH) to screen the genetic aberrations of VA. SUBJECTS AND METHODS: To identify candidate genes that might be causally involved in the pathogenesis of VA, genomic deoxyribonucleic acids were extracted from whole blood of 28 patients with VA who presented at Department of Cardiology at Seoul St. Mary's Hospital, Seoul, Korea. The copy number profiles of these patients was then analyzed using array CGH and reverse transcriptase (RT) quantitative polymerase chain reaction (PCR). RESULTS: Array CGH revealed gains in 31 different regions, with losses in the 4q35.2, 7q22.1, 10q26.3, 15q11.2, 16p13.11, 17p11.2 and 19q13.3 regions (more than 32% aberration in these regions). Several loci were found to be frequently including gains of 5p and 11q (50% of samples). The most common losses were found in 7q (54% of samples). Copy number aberrations in chromosomal regions were detected and corresponding genes were confirmed by RT quantitative PCR. The fold change levels were highest in the CTDP1 (18q23), HDAC10 (22q13.33), KCNQ1 (11p15.5-p15.4), NINJ2 (12p13.33), NOTCH2 (1p12-p11.2), PCSK6 (15q26.3), SDHA (5p15.33), and MUC17 (7q22.1) genes. CONCLUSION: Many candidate chromosomal regions that might be related to the pathogenesis of VA were detected by array CGH and should be systematically investigated to establish the causative and specific genes for VA.
Cardiology ; Coat Protein Complex I ; Comparative Genomic Hybridization ; Coronary Artery Disease ; Coronary Vessels ; DNA ; Humans ; Korea ; Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; Spasm

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.
Anti-Bacterial Agents ; Blotting, Northern ; Ciprofloxacin ; DNA Topoisomerases, Type I ; Escherichia* ; Fluoroquinolones* ; Point Mutation ; Polymerase Chain Reaction ; Quinolones ; Real-Time Polymerase Chain Reaction* ; RNA, Messenger ; Sequence Analysis, DNA

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.
Anti-Bacterial Agents ; Blotting, Northern ; Ciprofloxacin ; DNA Topoisomerases, Type I ; Escherichia* ; Fluoroquinolones* ; Point Mutation ; Polymerase Chain Reaction ; Quinolones ; Real-Time Polymerase Chain Reaction* ; RNA, Messenger ; Sequence Analysis, DNA

BACKGROUND: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in beta subunit of RNA polymerase is main mechanism of resistance. METHOD: In this study, rpoB gene for the ~3 subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. RESULTS: All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S53 IL) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not comfirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513-515 were new identified mutations for the first time. CONCLUSION: Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.
Base Pairing ; Codon ; DNA-Directed RNA Polymerases ; Drug Resistance, Multiple ; Early Diagnosis ; Isoniazid ; Mycobacterium tuberculosis ; Point Mutation ; Rifampin ; RNA Polymerase I ; Tuberculosis*