OBJECTIVE: The purpose of this study was to investigate the methods for analysis of restriction fragment length polymorphisms of hemophilia B (coagulation factorIX) gene in Korean population. METHODS: Genomic DNAs were extracted from 40 Korean females. In order to amplify genomic DNAs at the region of the polymorphic sites, two sets of primers (Hha I and Dde I) were synthesized. The primers were named as FIX1, FIX2 for Hha I, and Dde I 59, Dde I 39 for Dde I, respectively. Hha I primers annealed 3'-flanking region of the FactorIX gene and amplified 230 bp long fragment. The PCR fragment (230 bp) treated with Hha I endonuclease produced two fragments (150 bp and 80 bp), when the polymorphic site existed. Dde I primers annealed the region of the first intron of Factor IX gene and amplified 319 bp long fragments. People cases with Dde I polymorphic site are supposed to produce 369 bp long fragment. Results: It has been found that seven (14 X chromosomes) out of forty individuals showed Hha I polymorphism. However, none of the experimental People cases showed the Dde I polymorphism. CONCLUSIONS: By the analysis of 80 chromosomes, the PICs calculated from allele frequency of Hha I-RFLP (0.175/0.825) and that of Dde I-RFLP (0.0/1.0) were 0.289=[1-(0.1752+0.8252)] and 0=[1-(02+12)], respectively. From these results, it can be postulated that Hha I and Dde I polymorphisms of the Factor IX gene in Korean exhibited different patterns from those of Caucasian.
Blood Coagulation Factors* ; Blood Coagulation* ; Dichlorodiphenyl Dichloroethylene ; DNA ; Factor IX* ; Female ; Gene Frequency ; Genes, vif* ; Hemophilia B ; Humans ; Introns ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length*

PURPOSE: We hypothesize that bacillus Calmette-Guerin (BCG) may act through the regulation of various isoforms of nitric oxide synthase (NOS) [inducible (iNOS), endothelial (eNOS), and neuronal (nNOS)] genes and proteins expressions in rat bladder. MATERIALS AND METHODS: Adult female Sprague Dawley rats (200-250g) were injected into the urinary bladder transurethrally with BCG (22 rats) or saline (22 control rats) and after 2, 4, 6, and 12 hrs, and 1, 2, 3, 5, 7, 10 and 14 days, the bladders were harvested. Normal and BCG-treated rat bladders were analyzed for mRNA expressions for iNOS, eNOS, and nNOS by reverse transcriptase-polymerase chain reaction (RT- PCR). Protein expressions were determined by Western blotting analysis and immunohistochemistry. RESULTS: mRNA expression for iNOS was induced after 2 hrs of BCG injection in the rat bladder. Gene expression for iNOS was highest at 6 hrs and followed by decreased expression from 1 day, reaching its lowest level at 5 days. eNOS mRNA expression was detected in control bladders but its level was higher in the BCG-treated animals. nNOS mRNA expression was present in all the samples but did not change after BCG treatment. Western blotting analysis confirmed these findings. Immunohistochemical analysis demonstrated that eNOS was present mainly in endothelium, while iNOS was detected in stroma and inflammatory cells, and nNOS in epithelium and smooth muscle of rat bladder. CONCLUSIONS: The present study demonstrates that BCG treatment up-regulates gene and protein expressions of iNOS and eNOS in rat bladders, suggesting that BCG action may be mediated through NOS pathways.
Adult ; Animals ; Bacillus* ; Blotting, Western ; Endothelium ; Epithelium ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Muscle, Smooth ; Mycobacterium bovis ; Neurons ; Nitric Oxide Synthase* ; Nitric Oxide* ; Protein Isoforms ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Urinary Bladder*