No abstract available.
Digoxigenin* ; DNA* ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis*

BACKGROUND: It has been well known that bronchial asthma is a chronic inflammatory disorder. The "activation" of lymphocytes has a significant role in the pathogenesis of bronchial asthma. Among these lymphocytes, TH2-like rather than TH1-like lymphoytes are activated in the bronchial tissues from patients with atopic bronchial asthma. However, the difference of cytokines expression is not well documented between the atopic normal subjects and atopic asthmatics. METHODS: Bronchial tissues were obtained from the tweleve atopic and non-atpoic asthmatics and tweleve atopic and non-atopic healthy subjects for in stiu hybridizatin of IL-2, IL-4, IL-5, and INF-gamma. The probe of cytokines were tagged with digoxigenin by random priming method. RESULTS: The infiltration of many inflammatory cells on submucosa and denuded epithelium were observed in the bronchial tissue from patients with bronchial asthma. The RNase-treated bronchial tissues did not have the brown signal on the tissue, but, RNase-untreated bronchial tissues had the positive brown signal on the inflammatory cells under the basement membrane. The IL-2 positive signals were detected in 2 cases, IFN-gamma in 1 case, IL-4 in 2 cases, IL-5 in 2 cases among 6 non-atopic healthy subjects. The atopic healthy subjects showed 1 case of positive signal of IL-2 and IFN-gamma, but did not show any signals of IL-4 and IL-5. The positive signals of IL-2 were detected in 4 cases among 6 atopic and 6 non-atopic asthmatics, 2 cases and 1 case of IFN-gamma respectively, 4 cases and 3 cases of IL-4 respectively, 4 cases and 3 cases of IL-5 respectively. CONCLUSION: The lymphocytes were activated in the bronchus of asthmatics. Among lymphocytes, TH2-like lymphocytes may be involved in the pathogenesis of bronchial asthma. However, futher study with immunohistochemical stain may be necessary for defining the source of cytokines, because of TH2-like lymphocytes were also activated in some atopic healthy subjects.
Asthma* ; Basement Membrane ; Bronchi ; Cytokines* ; Digoxigenin ; Epithelium ; Humans ; Interleukin-2 ; Interleukin-4 ; Interleukin-5 ; Lymphocytes

BACKGROUND: To investigate epidemiology of a specific strain, and evaluate correlation between Mycobacterium tuberculosis restriction fragment length polymorphism (RFLP) and antimicrobial susceptibility, we studied about Mycobacterium tuberculosis RFLP isolated from Taegu area. METHODS: From Oct. 1997 and Mar. 1999, we isolated 54 strains of M. tuberculosis from the patients visiting Catholic University of Taegu Hyosung, Taegu, Korea. We studied their drug susceptibility and analyzed the Pvu treated RFLP using digoxigenin labeled IS6110 probe. RESULTS: Fifty-three had more than 6 bands of RFLP and strains with 10 bands were predominant (15 strain). Cluster analysis reveals eleven distinct clusters showing 57.4% of clustered rate (31 strains from A to K) and 35 independent patterns showing 64.8% of the diversity rate at 70% similarity level. Cluster A was the largest group (7 strains) and the next was cluster B (5 strains). Most of the patients with cluster A lived in Taegu city (85.7%) and all of 2 cluster K patients lived in Euisung area. There was no correlation between RFLP pattern and antimicrobial susceptibility, but all two strains of cluster H were resistant to isoniazid. Strains of clustered were also prevalent in the people of middle class. CONCLUSIONS: Compared to the RFLP analysis in the developed countries, Korea disclosed lower rate of diversity and higher clustered patterns of M. tuberculosis. The clustered strains were also prevalent among the people of middle class.
Daegu* ; Developed Countries ; Digoxigenin ; Epidemiology ; Humans ; Isoniazid ; Korea ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Restriction Fragment Length* ; Tuberculosis

Polymerase chain reaction (PCR) provides a powerful technique for identifying viruses and studying the homology between viral nucleic acids. However, PCR assay has limitations in its susceptibility to contamination or to enzymatic inhibitors. In order to avoid problems related to nucleic acid amplification, efforts have been made to obtain specific hybridization assays, such as dot blot hybridization (DBH). DBH has higher specificity and lower sensitivity than PCR. The aims of the present study were to develop a sensitive and specific assay for the detection of ovine herpesvirus 2 (OvHV-2), a gamma herpesvirus. PCR/DBH assay for detecting OvHV-2 DNA was developed and evaluated for its sensitivity and specificity. OvHV-2 specific primer pairs, 755/556, were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 102 viral copies. For DBH, the amplified DNA with OvHV-2 specific primer pairs, 556/555, was labeled by the incorporation of digoxigenin (DIG). This DIGlabeled probe was capable of detecting 104 viral copies of purified OvHV-2 DNA by DBH. On the other hand, PCR/ DBH was more sensitive than either PCR or DBH and also very specific. The results showed that the sensitivity of PCR/DBH was higher and stronger than that of PCR and DBH alone. This PCR/DBH assay can be applied efficiently to confirm the presence of OvHV-2 virus on clinical samples and to differentiate specifically between OvHV-2 infection and other viral infections.
Digoxigenin ; DNA ; Hand ; Limit of Detection ; Nucleic Acids ; Polymerase Chain Reaction ; Sensitivity and Specificity

BACKGROUND: In renal transplantation, a good HLA-DR match Is associated with successive graft outcome. But due to a number of technical problems, reliable serological DR typing cannot always be obtained. To compare the serological DR typing with DRBI DNA typing, we tested 103 specimens that had been frozen after serological typing, by PCR-SSOP typing method. METHODS: Serological DR typing was performed by complement-dependent microlymphocytotoxicity technique using commercial antisera kits, and DNA gyp ins was performed by PCR-SSOP, using one of the methods recommended by 12th International Histocompatibility Workshop. DNA amplification was done by DRBAMP-A and DRBAMP-B primers, and hybridization by 18 oligonucleotides labelled with digoxigenin.. RESULTS: The concordance rate between serologic typing and DNA typing was 76.7%. Most (79.0%) of discordant results were due to serological blanks turning out to be definable antigens by DNA typing and these antigens consisted of mainly DR5 splits but none of DR1, DR2, or DR7. CONCLUSIONS: In spite of technical improvement, serological typing method often can not define the accurate HLA-DR type. It is thought that combining serological typing with DNA typing Is necessary to achieve a higher success rate of graft outcome.
Digoxigenin ; DNA ; DNA Fingerprinting ; Education ; Histocompatibility ; HLA-DR Antigens* ; Immune Sera ; Kidney Transplantation ; Oligonucleotides ; Transplants

Recent molecular and physiological studies suggested that at least two H/K-ATPase isozymes are expressed in the rat kidney, and two distinct isoforms(HK alpha 2a, 2b) are resulted from alternative splicing of the 5-end of HK alpha 2 in distal colon by sequence analysis. Northern analysis and in situ hybridization(ISH) were carried out to analyze the expression of HK alpha 2a. and HK alpha 2b, mRNAs in rat kidney according to the changes of K-diet. Isoform specific 32P-labeled cDNA(for Northern) or digoxigenin labeled cRNA(for ISH) probes were used. Northern analysis demonstrated that HK a z. mRNA is abundantly expressed in normal(group 1: normal diet 2W) renal cortex, modestly in normal outer medulla, and weakly in normal inner medulla. The potassium-deprived rats(group 2: K-free diet 1W, and group 4: K-free diet 2W) expressed 40N lower levels in cortex and 2-4 fold higher levels of HKalpha 2a. mRNA in outer and inner medulla compared to normal rat. The potassium loading rats after potassium-deprivation(group 3: normal diet 1W after K-free diet 1W, and group 5: normal diet 1W after K-free diet 2W showed almost normal levels of HK alpha 2a. mRNA. HK alpha 2b, mRNA was not detected in any tissues of groups. By ISH, mRNA for HK alpha 2, was detected in the thick ascending limb, distal convoluted tubule, and the entire collecting duct. All groups exhibited comparable cellular patterns of labeling. Signal intensity of group 2 and 4 was less in cortical collecting duct(CCD), especially principal cells and much higher in the inner stripe of the outer medullary collecting duct(OMCDi) and the proximal inner medullary collecting duct(IMCD) compared to group 1. Group 3 and 5 exhibited signal intensity of group l. These results indicate that chronic hypokalemia enhances expression of HK alpha 2a, gene in OMCDi and proximal IMCD, decreases in CCD, and restores at normal levels in potassium-loading after potassium-deprivation and suggest that this isoform plays an important role in potassium balance by these segments accordiog to the changes of K-diet.
Alternative Splicing ; Animals ; Colon ; Diet* ; Digoxigenin ; Extremities ; Hypokalemia ; In Situ Hybridization ; Isoenzymes ; Kidney ; Potassium ; Rats ; RNA, Messenger ; Sequence Analysis

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species of mycoplasmas are important pathogens that cause respiratory infection in laboratory animals and that are known to affect experimental results obtained with contaminated animals. The aim of the present study was to develop a sensitive and specific assay for the detection of mycoplasma species. To this end, we developed a polymerase chain reaction and dot blot hybridization assay (PCR/DBH) for detecting mycoplasma DNA and evaluated it for its sensitivity and specificity. Mycoplasma consensus primer pairs were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 10(2) pg of mycoplasma purified DNA. For DBH, the amplified DNA was labeled by incorporation of digoxigenin (DIG). This DIG-labeled probe was capable of detecting 10(4) pg of purified mycoplasma DNA by DBH. PCR/DBH was more sensitive than PCR or DBH alone and was also very specific. Our PCR/DBH assay can be applied efficiently to confirm the presence of mycoplasma species on clinical samples and to differentiate between mycoplasma species infection and other bacterial infections.
Animals ; Animals, Laboratory ; Bacteria ; Bacterial Infections ; Chimera ; Consensus ; Digoxigenin ; DNA ; Limit of Detection ; Mycoplasma ; Polymerase Chain Reaction ; Sensitivity and Specificity

BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.
Autopsy ; Digoxigenin* ; Endopeptidase K ; Formaldehyde ; In Situ Hybridization* ; Paraffin ; Pathology ; Polymerase Chain Reaction ; Ribonucleases ; RNA Probes ; RNA* ; RNA, Messenger

Chronic hypokalemia alters Na+-K+-ATPase gene expression in several tissues. While it is established that Na+-K+-ATPase activity and alpha1 and beta1 subunit protein levels increase during K depletion in the outer medullary collecting duct (OMCD) and do not significantly change in the cortical collecting duct (CCD), little is known about the adaptive responses of the other isoforms in these other nephron segments. Accordingly, this study was performed to characterize the relative levels of expression and cellular distribution of mRNAs encoding the Na+-K+-ATPase subunit isoforms in normal and K-deprived (2 weeks) rats using the Northern analysis and in situ hybridization (ISH). Isoform specific 32P-labeled cDNA (for Northerns) or digoxigenin labeled cRNA (for ISH) probes were used. In normal rats, the order of expression amounts of all isoforms mRNAs from highest was outer medulla > cortex > inner medulla, and that of K-deprived rats was outer medulla > inner medulla > cortex. alpha1 mRNA levels were much greater than those of alpha2 or alpha3 in cortex, outer and inner medulla. mRNA levels for all isoforms were 2~3 folds greater in inner medulla of K-deprived rats compared to controls. In contrasts, the levels of all isoforms mRNAs in cortex and outer medulla were comparable between the two gruops. By ISH, mRNAs for all isoforms were observed in the S3 segment of proximal tubule, the cortical thick ascending limb (CTAL), medullary thick ascending limb (MTAL), distal convoluted tubule (DCT), connecting tuble (CNT), and the entire collecting duct. Both groups exhibited comparable cellular patterns of labeling, but the signal intensity of K-deprived rats was much greater in the proximal portion of the inner stripe of outer medullary collecting duct (OMCDi) and proximal portion of the inner medullary collecting duct (IMCD), and less in the MTAL compared to controls. The signal intensity of alpha1, alpha3, and beta1 isoforms was less in the CTAL, DCT, and CCD of K-deprived rats, but alpha2 isoform was slightly increased. These results suggest that chronic hypokalemia enhances expression of Na+-K+-ATPase subunit isoforms in the proximal portion of OMCDi and proximal IMCD, but not other nephron segments, and that these isoforms may participate in potassium conservation by these segments during potassium deprivation.
Animals ; Digoxigenin ; DNA, Complementary ; Extremities ; Gene Expression ; Hypokalemia* ; In Situ Hybridization ; Kidney ; Nephrons ; Potassium ; Protein Isoforms* ; Rats ; RNA, Complementary ; RNA, Messenger

BACKGROUND AND OBJECTIVES: MUC5AC is known to be a major secretory mucin in goblet cells of the mucosa of human lower respiratory tract. But in our preliminary study, we found that the levels of MUC8 mRNAs were significantly increased in the biopsy specimens of the nasal polyps whereas other mucin genes were not. This suggests the possibility that MUC8 might be one of the major overexpressed mucins in the nasal polyps. The purpose of this study is to investigate the cellular location of MUC5AC and MUC8 mRNA. Material and methods : Normal posterior ethmoid mucosa and the polyp tissue were fixed in 4% paraformaldehyde and were hybridized with the RNA riboprobe for MUC8 and the oligonucleotide probe for MUC5AC in the presence of digoxigenin (DIG). RESULT: In the normal posterior ethmoid mucosa, MUC 5AC mRNA and MUC8 mRNA were barely expressed in the epithelium and the submucosal glands. In the polyp epithelium, the expression of MUC 5AC mRNA was localized in the cytoplasm of goblet cells and the expression of MUC8 mRNA was strongly localized in the nucelus of the goblet cells, and weakly localized in the cytoplasm of the goblet cells. MUC8 mRNA was also expressed in low levels in the nucleus of the submucosal glands. CONCLUSION: MUC8 mRNA is localized mainly in the nucleus of goblet cells and is one of the major mucin genes overexpressed in goblet cells of thnasal polyp.
Biopsy ; Cytoplasm ; Digoxigenin ; Epithelium ; Goblet Cells ; Humans* ; Mucins ; Mucous Membrane ; Nasal Mucosa* ; Nasal Polyps ; Polyps ; Respiratory System ; RNA ; RNA, Messenger*