BACKGROUND: Aminophylline (theophylline) is one of the most frequent asthma therapies in Indonesia, although it remains as a narrow therapy. The effects of drugs are individualized and strongly influenced by genetic, one of which is CYP1A2 gene polymorphisms. This study aimed to determine the profile of CYP1A2 polymorphism and theophylline level in asthma exacerbation patients receiving intravenous aminophylline therapy.METHODS: This cross sectional study was conducted in the emergency room (ER), to adults asthma exacerbation patients without complication (n=27), visiting the ER. The gene polymorphism data were compared with theophylline levels in the blood using chi-square test.RESULTS: In the CYP1A2 gene polymorphism profile, the most common heterozygous alleles are T/G genotype of CYP1A2*1E and C/A genotype of CYP1A2*1F. Most homozygote alleles exist in CYP1A2*1D and CYP1A2*1F. There was significant difference between CYP1A2*1D (p<0.005), CYP1A2*1E (p<0.023) and CYP1A2*1F (p<0.000) polymorphisms and theophylline level.CONCLUSION: CYP1A2*1D, CYP1A2*1E and CYP1A2*1F gene polymorphisms had an effect on theophylline levels. However, no one experienced an overdose theophylline, and no correlation between theophylline levels with CYP1A2 gene polymorphism.KEYWORDS: exacerbation asthma, intravenous aminophylline, CYP1A2 polymorphism gene, theophylline
The effort of cloning the Lister strain of Salmonella typhi (NCTC 786, BCC 712) carB gene using pET-16b expression vector and E. coli JM109 as host cell has been done. The carB gene and the pET-16b expression vector were both prepared from their recombinant plasmid digested using BamHI and NdeI as restriction enzymes. The pG-carB-11-ST recombinant plasmid was isolated from Escherichia coli XL10(pG-carB-11-ST) and the pET-carA-ST recombinant plasmid was from Escherichia coli DH5α(pET-carA-ST). After being ligated (in a ratio of vector:gene of 1:3, 1:5 and 1:6) in the presence of T4 Bacteriofage DNA Ligase, the ligation mixture was used to transform Escherichia coli JM109 cells and plated out onto Luria Bertani medium containing ampicillin. An amount of 369 produced colonies were screened for the presence of the appropriate recombinant plasmid using combination of plasmid miniprep and agarose gel electrophoresis. None of the recombinant plasmids being suspected to carry the Salmonella typhi carB gene. It is suggested to repeat cloning process using E. coli JM109 or using E. coli XL10 as host cell which were known to have large cloning efficiency and can be used for large plasmids up to 25 kb.
Abstract–Biofilms play an important role in the distribution and pathogenicity of bacteria. In our previous research, we demonstrated that our isolated Bacillus sp. (collection of Microbiology Lab, Universitas Surabaya) exhibited in vitro antibiofilm activity. Bacilli generally produce cellulase, which is one of the enzymes responsible for bacterial biofilm degradation. Based on this fact, this study aimed (1) to determine whether or not the crude extract of our Bacillus sp. isolate is able to degrade biofilms formed by Escherichia coli, Staphylococcus aureus, and water-crane bacteria, and (2) to determine whether such crude extract contains active cellulase. The research steps began with the preparation of Bacillus sp. crude extract, biofilm degradation assay, cellulase activity assay on CMC Agar, and cellulase detection by SDS-PAGE and electrophoresis zymogram. The crude extract of Bacillus sp. effectively degraded the biofilm 30 min after contact. The crude extract decreased biofilms of Escherichia coli 23,56%, Staphylococcus aureus 27,78%, and water-crane bacteria 37,98%. Based on the zymogram result, it was shown that the crude extract contained active cellulase, with a size of approximately 35 kDA. It was concluded that the crude extract of Bacillus sp. has potential as an anti-biofilm agent and exhibits active cellulase activity. Keywords: Bacillus sp., crude extract, biofilm, SDS-PAGE, zymogram Abstrak–Biofilm mempunyai peran penting dalam penyebaran dan sifat patogenitas bakteri patogen. Pada penelitian sebelumnya telah dibuktikan bahwa isolat Bacillus sp. (koleksi Laboratorium Mikrobiologi, Universitas Surabaya) mempunyai aktivitas anti-biofilm secara in vitro. Bacillus umumnya menghasilkan selulase sedangkan selulase diketahui dapat meluruhkan biofilm bakteri. Berdasarkan hal di atas, penelitian ini bertujuan (1) untuk mengetahui apakah ekstrak kasar isolat Bacillus sp. ini dapat meluruhkan biofilm Escherichia coli dan Staphylococcus aureus, dan bakteri air keran; dan (2) untuk mengetahui apakah dalam ekstrak kasar terdapat selulase. Metode penelitian ini meliputi penyiapan ekstrak kasar sel Bacillus sp., pengujian aktifitas meluruhkan biofilm, uji aktivitas selulase pada Agar CMC, dan analisis keberadaan aktivitas selulase pada ekstrak kasar Bacillus sp. menggunakan SDS-PAGE dan elektroforesis zymogram. Hasil penelitian menunjukkan bahwa ektrak kasar isolat Bacillus sp. bekerja paling baik pada perlakuan 30 menit setelah kontak dengan biofilm bakteri. Pemberian ekstrak kasar dapat menurunkan biofilm Escherichia coli sebesar 23,56%; dan biofilm bakteri Staphylococcus aureus sebesar 27,78%, dan biofilm bakteri air keran sebesar 37,98%. Berdasarkan hasil zymogram, telihat bahwa ekstrak kasar isolat Bacillus sp. memiliki selulase aktif dengan ukuran sekitar 35 kDa. Dapat disimpulkan bahwa ekstrak kasar isolat Bacillus sp. berpotensi sebagai agen peluruh biofilm dan memiliki selulase aktif. Kata kunci: Bacillus sp., ekstrak kasar, biofilm, SDS-PAGE, zymogram
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