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"ABSTRACT

Cytochrome P450 isoform 2C9 (CYP2C9) is a main enzyme which metabolizes phenytoin [I]. The inhibition of this enzyme will increase plasma level of phenytoin. Cimetidine is known as drug that inhibits this enzyme, resulting an increased plasma level of phenytoin [2]. Recently, the three dimentional molecular basis of interaction between phenytoin and cimetidine toward CYP2C9 has not been described well yet. The present findings may represent an important advance for understanding interaction CYP2C9 with drugs to predict its toxicity an also metabolism based on structural interaction from docking results. A computational methodology, molecular docking can be used to analyze interaction which exist between ligand and macromolecule. AutoDock is one of the most commonly used methodology, shows the efficiency of scoring function ligand that bound to its active site [3]. So that, it can be used to understand about interaction between phenytoin and cimetidine in CYP2C9. Crystal structure of CYP2C9 complexed with flurbiprofen (PDB ID: 1R90) has resolution 2.00 A. This structure, used in this experiment, has the closed conformational structure and complexed with S-warfarin. Three dimensional structure of phenytoin and cimetidine were minimized, charge were added for docking preparation. Binding of substrate phenytoin in CYP2C9 is stabilized by hidrogen bonds, interaction with cationic residue Argl08, hydrophobic interaction particularly with Phel 14. On the other side, binding of cimetidine inhibitor in CYP2C9 is stabilized by hydrogen bonds with some amino acid residues, including Glu300 which has role as anionic residue, also the exist of hydrophobic interaction. Cimetidine being competitive inhibitor of CYP2C9 at the substrate recognition site of phenytoin. "

"Cytochrome P450 3A4 (CYP3A4) contributes to the metabolism of 50% of drugs used in therapy [l]. Nowadays, the structures of CYP3A4 are available through crystallography technique and these structures show that CYP3A4 has a flexible active site which allows many probabilities of ligand interaction [2]. Nelfinavir, one of the HIV-Protease inhibitors, is a substrate and also an inhibitor of CYP3A4. However, the CYP3A4-mediated metabolism of nelfinavir result in unknown reactive metabolites which then inactivate CYP3A4 [3]. In silica method through molecular docking is used in this research to study the inhibition of CYP3A4 by nelfinavir and to predict the reactive metabolites of nelfinavir that inactivate CYP3A4. The docking result show that nelfinavir fits the CYP3A4 active site with the conformation that coordinates to the forming of M8 metabolite of nelfinavir. The docking result for M8 gives positive binding energy and the docking result for the intermediate metabolite between nelfinavir and M8 indicates that this intermediate metabolite is responsible for the inhibition of CYP3A4 by nelfinavir through mechanism-based inactivation."

"Pemalsuan kandungan daging merupakan sebuah masalah yang besar, terlebih di negara dengan mayoritas penduduknya beragama Islam seperti Indonesia. Sejauh ini, proses uji deteksi kehalalan berpedoman pada rekomendasi ISO yang memanfaatkan qPCR dengan gen target ACTB pada babi, namun komparasi antara reliabilitas gen ACTB dan reliabilitas gen lainnya belum banyak diuji. Gen alternatif yang dinilai berpotensi untuk dijadikan gen target pada primer untuk uji deteksi halal adalah gen yang terdapat pada DNA mitokondria (mtDNA), yaitu COI, Cytb, dan 12S rRNA. Spesifisitas ketiga primer tersebut sudah teruji secara in silico sebesar 100%. Namun, spesifisitas in silico tidak selalu menunjukkan hasil yang sama secara in vitro, sehingga spesifisitas ketiga primer perlu diuji secara in vitro dan dibandingkan dengan primer ACTB terhadap DNA daging babi hutan dan babi domestik. Metode yang digunakan dalam penelitian ini terdiri atas isolasi DNA dari daging babi domestik, babi hutan, sapi, kambing, ayam, ikan tuna, dan ikan mas, kuantifikasi DNA, optimasi suhu annealing primer, dan uji spesifisitas melalui qPCR. Konsentrasi DNA yang diisolasi berkisar dari 21,0 ng/μL hingga 155,5 ng/μL, sedangkan kemurnian DNA berkisar dari 1,393 hingga 1,929. Suhu annealing optimal yang dipilih adalah 57oC untuk ketiga primer. Berdasarkan hasil qPCR, spesifisitas primer gen COI, Cytb, dan 12S rRNA masing-masing sebesar 50%, 25%, dan 50%, dan berada di bawah spesifisitas ACTB sebesar 100%, kemungkinan diakibatkan oleh proses desain yang kurang optimal. Oleh karena itu, disimpulkan bahwa spesifisitas primer COI, Cytb, dan 12S rRNA masih tergolong rendah dan perlu dilakukan upaya desain ulang primer untuk meningkatkan spesifisitasnya.

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Meat content adulteration is a prominent problem in a predominantly Muslim country such as Indonesia. To this date, the majority of halal detection activities have followed the recommendation given by ISO, utilizing qPCR and the target gene of ACTB in pigs, but its’ reliability is yet to be compared with other genes intensively. Mitochondrial DNA (mtDNA) genes, such as COI, Cytb, and 12S rRNA are considered worthy alternatives of becoming primer target genes in halal detection, with satisfactory in silico specificity of 100%. Alas, in silico specificity does not always correspond to in vitro results. Therefore, the three primers’ specificities towards wild boar and domesticated pig DNA need to be evaluated in vitro and compared with the ACTB primer. The methods used in this research include DNA isolation from pork and wild boar meat, beef, goat meat, chicken meat, tuna, and carp meat, DNA quantification, primer annealing temperature optimization, and specificity evaluation through qPCR. Isolated DNA concentrations range from 21,0 ng/μL to 155,5 ng/μL, while DNA purity ranges from 1,393 to 1,929. The optimal annealing temperature chosen for the three primers is 57oC. Based on the qPCR results, the specificities for primers COI, Cytb, and 12S rRNA are 50%, 25%, and 50%, and are below ACTB primers’ specificity which stands at 100%, most likely due to suboptimal primer design. Hence, it is concluded that the specificities of COI, Cytb, and 12S rRNA primers are still considered low, and redesigning is necessary for improval."