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AMPLIFIKASI GEN PENYANDI PROTEIN FUSION VIRUS TETELO DARI SPESIMEN LAPANGAN DENGAN METODE ONESTEP RT-PCR. (AMPIFICATION OF FUSION PROTEIN ENCODING GENE OF NEWCASTLE DISEASE VIRUS FROM FIELD SPECIMENS BY ONESTEP RT-PCR METHOD) Haryanto, Aris; Kristiawan, David; Irianingsih, Sri Handayani; Yudianingtyas, Dini Wahyu
Jurnal Veteriner Vol 14 No 3 (2013)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Tetelo or Newcastle Disease (ND)  virus is belong to the family Paramyxoviridae, which has a singlestranded RNA (ss RNA) genome and it has viral envelope.  The viral envelope consists of two majorproteins, namely Haemagglutinin/Neuraminidase (H/N) and Fusion (F) protein. Molecular diagnosticmethods OneStep RT-PCR is a commonly diagnostic tool that used to diagnose of Tetelo in poultry. In thisstudy the diagnosis of Tetelo is accomplished by amplification of F protein encoding gene of Tetelo virusdirectly from field specimens without inoculation and propagation of Tetelo virus into embryonated chickeneggs. The objective of this study is to conduct rapid diagnosis of Tetelo virus directly from the field specimensbased on the amplification of the F gene by a OneStep RT-PCR method, so that the results of this study canbe used to assist in the identification of Tetelo virus directly from the field specimens. The results showedthat from the 15 samples of virus which isolated from tracheal swabs of clinical poultry showing symptomsof Tetelo virus infection, a total of 12 samples from 15 tested sampels (80%) are positive tested for Tetelovirus infection. They were indicated by the amplification product of DNA fragments in size of 362 bp.OneStep RT-PCR is a method for rapid and effective diagnosis which can be used  to diagnose of Tetelovirus directly from field specimens.
PREPARATION OF LYMPHOCYTE CULTURE CELL FROM PERIPHERIAL BLOOD OF NASOPHARYNGEAL CARCINOMA PATIENTS Haryanto, Aris; Mubarika, Sofia; Wijayanti, Nastiti
Jurnal Sain Veteriner Vol 18, No 1&2 (2000)
Publisher : Fakultas Kedokteran Hewan Universitas Gadjah Mada bekerjasama dengan PB PDHI

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/jsv.8239

Abstract

Lymphocyte is leukocyte component that difficult to culture in vitro. Several viruses need lymphocytes as host cell in order to proliferate and growth in this media such as Epstein-Barr virus (EBV). This virus was associated with malignant disease like nasopharyngeal carcinoma (NPC). The objectives of this research are to develop and to prepare lymphocyte cell culture as material source of DNA for molecular analysis of virus. Peripheral blood was collected from NPC patients which is histopatologically and serologically positive of EBV. Lymphocytes were separated from the other blood components using ficcol-histopaque. Lymphocytes were diluted using RPMt medium then they were cultivated into 96 microwell plate with concentration of 106 cell/ml. The medium consist of 10% FBS, RPMI, Penstrep and FK 506. Culture of lymphocytes were incubated in 5% CO2 at 37°C. The lymphocyte cultures developed and grew confluendy during the first week. Only B cells whith EBV would be well establish. After 50 cell generations, lymphocytes were transformed and immortalized to be lymphoblastoid cell fine (LCL).
Effect of the HBV Capsid Assembly Inhibitor Bayer 41-4109 on the Intracellular Localization of EGFP-Core Fusion Proteins Haryanto, Aris; Wijayanti, Nastiti; Kann, Michael
Indonesian Journal of Biotechnology Vol 12, No 2 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Bayer 41-4109 is heteroarylpyrimidine (HAP) which has been identified as potent of HBV capsid assemblyinhibitor. The present study was to study effect of Bayer 41-4109 treatment on the intracellular localization ofEGFP-Core fusion proteins into HepG2 cells. Three recombinant plasmids of pEGFP-Core with single, double andtriple NLS of HBV core (EGFP-Core 1C, 2C and 3C ) and two recombinant plasmids with single and triple NLS ofSV-40 (EGFP-Core 1 and 3 SV-40) were used in this work. After transient transfected into HepG2 cells and treatedwith Bayer 41-4109, the intracellular localization of expressed fusion proteins from all plasmid constructions weredetermined and quantified under confocal laser microscope. Results shown that Bayer 41-4109 treatment in HepG2cells inhibited the nuclear localization of EGFP-Core with single of triple HBV core NLS. As well as the constructionsof expressed fusion protein with single and triple SV-40 NLS (EGFP-Core 1 and 3 SV-40 NLS) showeddecreasing the nuclear localization after treated with Bayer 41-4109, even not as strong as EGFP-Core 1C and 3CNLS. Bayer 41-4109 has been identified as a potent inhibitors of HBV replication which has multiple effects on HBVcapsid assembly. It may inhibit virus replication by inducing assembly inappropriately and by misdirectingassembly decreasing the stability of normal capsids.Keywords: HBV capsid, Bayer 41-4109, EGFP-Core fusion protein, HepG2 cell
Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell) Haryanto, Aris; Kann, Michael
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization
Effect of Nuclear Export Inhibitor Leptomycin B on the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells Haryanto, Aris; Wijayanti, Nastiti; Kann, Michael
Indonesian Journal of Biotechnology Vol 15, No 2 (2010)
Publisher : Universitas Gadjah Mada

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Abstract

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of manyregulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants.In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cell
Molecular Genotyping of HBV by using Nested PCR-RFLP among Hepatitis B Patients in Daerah Istimewa Yogyakarta Province and Surrounding Area Haryanto, Aris; Mulyani, Nenny Sri; Widowati, Titis; Wijayanti, Nastiti; Hadi, Purnomo
Indonesian Journal of Biotechnology Vol 13, No 2 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

Hepatitis B virus (HBV) can be classified into 8 genotypes, genotype A to H. Genotype of HBV is important for clinical and etiological investigations. Research for HBV genotyping, HBV transmission study using nested PCR and HBV genotyping based on RFLP using restriction enzymes have been reported. However, both of those methods have been not applied for HBV genotyping study among hepatitis B patients in endemic area, like Indonesia yet. Molecular genotyping of HBV will describe epidemiology, pathogenesis and clinical implication of HBV. Combination of nested PCR and RFLP (nested PCR-RFLP) method to determine HBV genotype in Indonesia is still less information. The objectives of study were to develop a system for HBV genotyping by nested PCR combined with RFLP (nested PCR-RFLP) method based on nucleotide sequence of surface protein encoding</div><div>gene (S gene) in HBV genome and to confirm HBV genotypes which predominantly found among hepatitis B patients in Daerah Istimewa Yogyakarta Province and surrounding area. Total of 149 sera from chronic hepatitis B patients from Daerah Istimewa Yogyakarta and surrounding areas were collected for in this work. Viral DNA were extracted from sera of hepatitis B patients and used as template for first round nested PCR amplification using outer primers set. Amplicons of first round PCR were used as template for second round amplification using inner primers set. Then, amplicons of second round nested PCR were restriction digested by Sty I and Bsr I enzymes. For HBV genotyping then the restriction products were analyzed by RFLP based on restriction pattern. Results showed that the first round nested PCR amplification generated DNA fragments of whole S gene in length 1.233 bp, and in second round nested PCR amplification using inner primer set generated DNA fragments 585 bp in length. Genotype analysis for all samples using nested PCR-RFLP methods by restriction digested of Sty I and Bsr I enzymes found only 2 HBV genotypes among hepatitis B patients, namely genotype B and C. Quantification</div><div>data showed that most of hepatitis B patients found infected by HBV genotype B (92,8%), genotype C (3,6%) and unidentified genotype (3,6%). Nested PCR-RFLP methods for HBV genotyping is simple and inexpensive for clinical diagnostic in large scale.
Effect of Staurosporine on the Intracellular Localization of Hepatitis B Virus Core Protein Haryanto, Aris; Wijayanti, Nastiti; Kann, Michael
Indonesian Journal of Biotechnology Vol 12, No 1 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

protein is also including in the HBV genome targeting into the nucleus through modulating carboxyl residues byphosphorylation. Nuclear localication Signal (NLS) in HBV core protein is inside the virion structure and it must beunmasked in order to function, perhaps by phosphorylation. Phosphorylation of of HBV core protein in turn couldbegin to alter capsid conformation. Staurosporine is a natural product originally isolated from bacteriumStreptomyces staurosporeus. Staurosporine was discovered to have biological activities ranging from anti-fungal toanti-hypertensive. The interest in these activities resulted in a large investigative effort in chemistry and biology andthe discovery of the potential for anti-cancer treatment. The main biological activity of Staurosporine is the inhibitionof protein kinases through the prevention of ATP binding to the kinase. In the present study, we have studied theintracellular localization of EGFP-Core fusion protein with triple HBV core and SV-40 nuclear localization signal atits carboxyl terminal in presence and absence of Staurosporine. We also to study the effect of Staurosporine treatmenton the intracellular localization of EGFP-Core fusion protein in the hepatocyte cells line of HepG2 cell. Resultsshowed that effect of Staurosporine is prevent the nuclear localization of EGFP-Core fusion protein into nucleusthrough an inhibition of the phosphorylation of core protein. Stauroporine also prevents cell division so that passivetrapping of core protein is inhibited.
Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR Wijayanti, Nastiti; Wibawa, Tri; Nirwati, Hera; Haryanto, Aris; S, Sutaryo1
Indonesian Journal of Biotechnology Vol 11, No 2 (2006)
Publisher : Universitas Gadjah Mada

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Abstract

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR
Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein Haryanto, Aris
Indonesian Journal of Biotechnology Vol 10, No 1 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.
NTRACELLULAR LOCALIZATION OF HBV CAPSID IN HEPATOCYTE LINE AFTER TRANSFECTED BY THE ENTIRE HBV GENOME = LOKALISASI INTRASELULER KAPSID HBV PADA SEL LINE HEPATOSIT SETELAH DITRANSFEKSI DENGAN GENOM UTUH HBV. Haryanto, Aris; Kann, Michael
Jurnal Sain Veteriner Vol 24, No 1 (2006): JUNI
Publisher : Fakultas Kedokteran Hewan Universitas Gadjah Mada bekerjasama dengan PB PDHI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (2750.247 KB) | DOI: 10.22146/jsv.339

Abstract

HBV replicates within the nucleus of hepatocyte using cellular transport machinery for the import of their genomes into the nucleus. Genome of HBV has to transported through the cytoplasm towards the nuclear pore complex (NPC) followed by subsequent passage through the pore. HBV capsid is involved in a number of important functions in the replication cycle of HBV. It can be detected in the nucleus, cytoplasm or both within infected hepatocytes. Nuclear localization of HBV capsid protein, which is karyophilic, depends on the cell cycle. The objective of the present study was to analyzes the intracellular localization of HBV capsid protein after transfected by entire HBV genome into hepatocyte cell lines (HuH-7) and to determine the predominantly localization of the capsid into cell compartment. In this work we analysed the intracellular localization of the HBV capsid in human hepatocyte cell lines liuH-7 by transfection using entire HBV genome and transient expression. The transfected cells were fixed and an indirect immune staining against the HBV capsid was performed to detect the capsid. To verify the location within the cell, an additional co-staining against the nuclear pore complexes was performed. The Intracellular localization of the HBV capsid and NPC were analyzed by a confocal laser scan microscope. The observed of localizations into the transfected cells were classified to be predominantly as nuclear localization, cytoplasmic localization or distributed within both of these compartments. Result of this study indicated that Staining of HBV capsid found predominantly within the nucleus (71%). Less frequently, the HBV capsid localized within the cytoplasm (26%). Only in a minority of cases, the capsids were localized within cytoplasm and nucleus (3%). This low frequency indicate that the capsids were not diffusing within the cells being in accordance to the in vivo situation in which the nuclear membrane was impermeable for the capsid.