HAK HOTTA
Center for Infectious Diseases, Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan

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Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of Mycobacterium tuberculosis in Indonesian clinical isolates Lisdawati, Vivi; Oshibe, Tomohiro; Tsuji, Hidetaka; Sudiro, Tjahjani M.; Adianti, Myrna; Sukarso, Triyani; Arief, Holy; Hotta, Hak; Sudarmono, Pratiwi
Medical Journal of Indonesia Vol 21, No 4 (2012): November
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (526.167 KB) | DOI: 10.13181/mji.v21i4.502

Abstract

Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as “a set” from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia.Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA’s Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system.Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate.Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia. (Med J Indones. 2012;21:188-95)Keywords: Loop-mediated isothermal amplification, rim gene, 16S rRNA gene, gyrB gene, Mycobacterium tuberculosis
Antiviral effect of Archidendron pauciflorum leaves extract to hepatitis C virus: An in vitro study in JFH-1 strain Hartati, Sri; Aoki, Chie; Hanafi, Muhammad; Angelina, Marissa; Soedarmono, Pratiwi; Hotta, Hak
Medical Journal of Indonesia Vol 27, No 1 (2018): March
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (497.399 KB) | DOI: 10.13181/mji.v27i1.2189

Abstract

Background: Hepatitis C virus (HCV) is a leading cause of chronic liver diseases. Drug resistance to the regimen is also increasing. Hence, there is a need for new anti-HCV agents that are less toxic and more efficacious. The aim of this study is to evaluate the  possibility of A. pauciflorum extracts can be a antiviral drug.Methods: Huh-7it cells were infected with the HCV genotype 2a strain JFH-I in the presence of methanol extracts of Archidenron pauciflorum. The methanol extract further partition used n-hexane, ethyl acetate, n-butanol, and water showed in which butanol extracts exerted the strongest IC50 (6.3 g/ml). Further, the butanol fraction was fractionated and yielded into 13 fractions.Results: The methanol extract of the leaves of A. pauciflorum exhibited concentration dependent inhibition against the JFH1 strain of HCV genotype 2a with an IC50 is 72.5 μg/ml. The butanol fraction exhibited the highest anti-HCV activity with an IC50 is 6.3 μg/ml. The butanol fraction was fractionated which yielded 13 fractions. Fractions 5 and 13 exhibited high anti-HCV activities with IC50 is 5.0 μg/ml and 8.5 μg/ml and a time-of-addition study demonstrated that fraction 5 inhibited viral infection at the post-entry step, whereas fraction 13 primarily inhibited the viral entry step.Conclusion: The extract A. pauciflorum can be used as a herbal-based antiviral drug.
Aktivitas Sitotoksisitas Ekstrak Metanol Daun Sirsak (Annona muricata L.) terhadap Karsinoma Hepatoseluler Strain HUH7IT-1 Cell Line Apriyanto, Dadan Ramadhan; Hartati, Sri; Dewi, Beti Ernawati; Aoki-Utsubo, Chie; Hotta, Hak
Tunas Medika Jurnal Kedokteran & Kesehatan Vol 4, No 1 (2018): Tunas Medika Jurnal Kedokteran & kesehatan
Publisher : Tunas Medika Jurnal Kedokteran & Kesehatan

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Abstract

ABSTRAKLatar Belakang: Karsinoma hepatoseluler (HCC) merupakan tumor ganas hati primer dengan prognosis pada umumnya dapat menyebabkan kematian. Studi awal penelitian antiviral hepatitis C pada tumbuhan Sirsak (Annona muricata L.) pada konsentrasi 20 μg/mL memperlihatkan toksisitas yang sangat tinggi terhadap Huh7it-1 cell line, yang diindikasi memiliki potensi anti kanker terhadap sel hati, sehingga penelitian ini bertujuan menguji beberapa konsentrasi lebih rendah pada ekstrak metanol daun Annona muricata L. (EMDAM) terhadap Karsinoma Hepatoseluler strain Huh7it-1 cell line.Metode: Sel diuji dengan konsentrasi 20, 10, 5, 2.5, 1.25, 0.6, 0.3 μg/mL selama 48 jam. Sitotoksisitas EMDAM terhadap Huh7it-1 dilihat dengan mikrokop inverted dan selanjutnya diukur dengan metode MTT [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].Hasil: Hasil uji menunjukkan sel memperlihatkan bentuk tidak monolayer pada mikroskop inverted dengan sitotoksisitas hingga konsentrasi terendah pada 0.3 μg/mL mencapai 84,7%, sehingga konsentrasi 50% Sitotoksisitas (CC50) < 0.3 μg/mL.Simpulan: Hasil uji mengindikasi bahwa EMDAM memiliki potential terhadap aktivitas anti kanker hati. Studi lebih lanjut diperlukan untuk purifikasi untuk senyawa aktif sebagai antikanker atau target mekanisme terhadap aktivitas anti kanker hati.Kata kunci: Karsinoma Hepatoseluler, Huh7it-1, Sitotoksisitas, Annona muricataABSTRACTBackground: Hepatocellular carcinoma (HCC) is a malignant tumor of liver cells with prognosis can cause death within 2-3 months. Previous studies of Annona muricata L. on anti-HCV studies at concentrations of 20 μg / mL showed very high toxicity to Huh7it-1 cell line, it was indicated to have anti-cancer potential of liver cells, so this study tested the potency of anticancer activity extract methanol leaf Annona muricata L. (EMDAM) against Hepatocellular Carcinoma Huh7it-1 strain cell line with low dose.Methods: Cells were tested with concentrations of 20, 10, 5, 2.5, 1.25, 0.6, 0.3 μg / mL for 48 hours. The EMDAM cytotoxicity of Huh7it-1 was seen with an inverted microcomputer and then measured with MTT assay [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].Results: The results showed that the cells presented non-monolayer form in an inverted microscope with cytotoxicity until the lowest concentration of 0.3 μg / mL reached 84.7%, thus concentrating 50% cytotoxicity (CC50) <0.3 μg / mL.Conclusion: The results indicate that EMDAM has the potential for anti-liver cancer activity. Further studies are needed for purification for active compounds as anticancer or target mechanisms against anti-liver cancer activity.Keywords: Hepatocellular carcinoma, Huh7it-1, Cytotoxicity, Annona muricata