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Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate Sulistyaningsih, Erma; Moeljopawiro, Sukarti; Subandono, Jarot; Artama, Wayan T.
Indonesian Journal of Biotechnology Vol 10, No 1 (2005)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (196.441 KB) | DOI: 10.22146/ijbiotech.7411

Abstract

Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA
Phylogenetic relationship of Gram Negative Bacteria of Enterobacteriaceae Family in the Positive Widal Blood Cultures based on 16S rRNA Gene Sequences Darmawati, Sri; Sembiring, Langkah; Asmara, Widya; Artama, Wayan T.; Kawaichi, Masashi
Indonesian Journal of Biotechnology Vol 19, No 1 (2014)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (431.864 KB)

Abstract

The purpose of this study was to analyze the phylogenetic relationship of Gram negative bacteria (3strains of Salmonella typhi, 1 strain of Escherichia coli, 1 strain of Serratia marcescens, and 3 strains of Enterobactercloacae) of Enterobacteriaceae family in positive Widal blood cultures based on 16S rRNA gene sequences. Theresults respectively showed that each two 16S rRNA gene clones of Serratia marcescens KD 08.4 had a closerelationship with 16S rRNA gene of Serrratia marcescens ATCC 13880 (similarity: 99.53-99.8%), Eschericia coliBA 30.1 with Eschericia coli ATCC 11775T (similarity: 99.38-99.67%), Salmonella typhi BA 07.4, Salmonella typhiKD 30.4, and Salmonella typhi SA 02.2 with Salmonella typhi ATCC 19430T (similarity: 99.4-100%) as well as theisolates of Enterobacter cloacae SA 02.1, Enterobacter cloacae BA 45.4.1, one 16S rRNA gene clone of Enterobactercloacae TG 03.5 with Enterobacter cloacae ATCC 23373 (similarity: 99.0-99.87%).
Analysis of Toxoplasma gondii Repeat Region 529 bp (NCBI Acc. No. AF146527) as a Probe Candidate for Molecular Diagnosis of Toxoplasmosis Pratama, Dyah Ayu Oktavianie A; ., Sumartono; Artama, Wayan T.
Indonesian Journal of Biotechnology Vol 14, No 1 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (670.229 KB)

Abstract

            Toxoplasmosis is a disease caused by protozoan parasite Toxoplasma gondii. The infection is commonly asymptomatic. The availability of confirmative and accurate detection system is really needed. This research was aimed to develop a molecular diagnosis based on the conserved and high copy number repeat region of Toxoplasma gondii with hibridization method. Nucleic acid was isolated from tachyzoites. The repeat region of T. gondii was amplified using PuRe Taq Ready To Go-PCR Beads (Amersham Bioscience),  forward primer 5’- GAC TCG GGC CCA GCT GCG  -3’ and reverse primer 5’- CCT CTC CTA CCC CTC CTC -3’. The amplicon was sequenced using ABI Prism 3100-Avant Genetic Analyzer (PT. Charoen Pokphand, Jakarta). Probe was labeled using digoxigenin-11-dUTP. Application of probe to detect it’s complementary nucloeic acid was done by hibridization method. The research concluded that probe toxo-103 bp was highly homolog with several strain of T. gondii and it has no homology either with host’s genome or other parasites which have close genetic relationship with T. gondii.   Hybridization analysis showed that probe could detect the complementary nucleic acid up to 10 ng/ul concentration.      
Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate Sulistyaningsih, Erma; Moeljopawiro, Sukarti; Subandono, Jarot; Artama, Wayan T.
Indonesian Journal of Biotechnology Vol 10, No 1 (2005)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (196.441 KB)

Abstract

Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA
Chemosystematic of Enterobacteriaceae Familia Obtained from Blood Cultures Based on Total Protein Profiles Darmawati, Sri; Sembiring, Langkah; Asmara, Widya; Artama, Wayan T.; Anwar, Syaiful
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (303.344 KB)

Abstract

The purpose of this study was to determine the chemosystematic of 14 strains of bacteria in blood cultures from Semarang using 1 reference strain S. typhi NCTC 786, based on the total protein profi les with the similarity relationship analysis based on Simple Matching Coeffi cient (SSM) analysis and algorithm methodof unweighted pair group with averages (UPGMA) presented in a dendrogram. The results showed that thechemosystematic based on the total protein profi les using SDS-PAGE method can classify the member ofbacterial strains of each species. The Clusters respectively consist of 4 strains of S. typhi (similarity: 89.7%),2 strains of Ser. marcescens (similarity: 89.7%), two strains of E. coli, and one strain of Salmonella ssp, S. typhi NCTC 786 (similarity: 100%). Those three incorporated clusters had the similarity value of 75.3%. Those four strains of Ent. cloacae composed in one cluster (similarity: 100%) are incorporated in a cluster consisting of one strain of Kleb. pneumoniae (similarity: 92.9%). Both clusters were incorporated in a cluster consisting of S. typhi NCTC 786 (similarity: 67.9%).Key words: Enterobacteriaceae, chemosystematic, blood cultures, protein profile
Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate Artama, Wayan T.; Sari, Yulia; Subekti, Didik Tulus; Poerwanto, Soenarwan Hery; Subandono, Jarot
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2
Analisis Sekuen Gen Tubulin-β Isotipe 1 Cacing Haemonchus contortus Isolat Resisten terhadap Benzimidazole pada Domba di Indonesia Haryuningtyas, Dyah; Artama, Wayan T.
Jurnal AgroBiogen Vol 4, No 2 (2008): Oktober
Publisher : Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

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Abstract

Benzimidazole (BZ)resistance to gastrointestinal nematodes in small ruminants(sheep and goat) has become a significant problem worldwide.Evidences of anthelmintic resistance to albendazole inIndonesia has been reported from some government ownedfarms in West Java, Central Java, and Yogyakarta. Previousstudy on the sheep parasite H. contortus had shown that theBZ resistance was related to selection for individuals in apopulation possesing a spesific β-tubulin isotype 1 gene. Thestudy is aimed to determine mutation on coding region ofcentral part of β-tubulin isotype 1 gene of H. contortus resistantstrain from Indonesia. Seven H. contortus worms wereisolated from four BZ resistant sheep from two governmentfarms (SPTD Trijaya, Kuningan, West Java, and UPTDPelayanan Kesehatan Hewan, Bantul, Yogyakarta), and froma BZ susceptible sheep from Cicurug, Sukabumi, West Java.DNA was extracted individually from female H. contortusworms. A fragment of 520 bp β-tubulin isotype 1 gene exon3, 4, 5 was amplified using the PCR technique and thensequenced. The results showed that a single mutationoccurred in codon 200 (from phenilalanine to tyrosine) hadcaused benzimidazole resistance in H. contortus from SPTDTrijaya, Kuningan, West Java. Mutation in β-tubulin isotype 1gene of H. contortus from UPTD Pelayanan KesehatanHewan, Yogyakarta, occurred in codon 198 (from glutamateto glycine), codon 201 (from cystein to stop codon), andcodon 202 (from isoleucyne to stop codon).
Directly observed treatment increases drug compliance in lymphatic filariasis mass drug administration Rosanti, Tutik Ida; Mardihusodo, Sugeng Juwono; Artama, Wayan T.
Universa Medicina Vol 35, No 2 (2016)
Publisher : Faculty of Medicine, Trisakti University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18051/UnivMed.2016.v35.119-127

Abstract

Backgroud. Mass drug administration (MDA) has been one of the strategies for lymphatic filariasis elimination. Since the start of implementation in 2011 in Pekalongan, no study on MDA acceptability has yet been done. The objectives of the study were to determine the microfilaria rate (mf rate) and the risk factors of drug compliance in the MDA program. MethodsA longitudinal study was conducted at Pabean region, Pekalongan City. There were 90 household heads as subjects, who were selected by proportional cluster random sampling. Microfilaria rate (mf rate) was determined by finger blood examination. Drug compliance was measured using questionnaires and observation sheets. Drug compliance observers, filariasis counseling participation, and presence of filariasis patients were factors influencing drug compliance. ResultsMf rate in 2015 was 1.35% and drug compliance rate was 86.80%. Reasons for failing to take drugs were fear of side effects (50%), refusals (25%), laziness (16.7%), and perceiving the drug to be useless (8.3%). The chi-square test shows a significant difference between the presence of drug compliance observer and compliance (p=0.006). Filariasis counseling participation and presence of filariasis patients did not show a significant difference with drug compliance (p= 0.986).ConclusionsFamilies as the source of observers was associated with increased filariasis drug compliance. It is therefore essential to address the issues linked to low compliance to make the program more efficient and achieve the goal of filariasis elimination.
Phylogenetic relationship of Gram Negative Bacteria of Enterobacteriaceae Family in the Positive Widal Blood Cultures based on 16S rRNA Gene Sequences Darmawati, Sri; Sembiring, Langkah; Asmara, Widya; Artama, Wayan T.; Kawaichi, Masashi
Indonesian Journal of Biotechnology Vol 19, No 1 (2014)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (431.864 KB) | DOI: 10.22146/ijbiotech.8635

Abstract

The purpose of this study was to analyze the phylogenetic relationship of Gram negative bacteria (3strains of Salmonella typhi, 1 strain of Escherichia coli, 1 strain of Serratia marcescens, and 3 strains of Enterobactercloacae) of Enterobacteriaceae family in positive Widal blood cultures based on 16S rRNA gene sequences. Theresults respectively showed that each two 16S rRNA gene clones of Serratia marcescens KD 08.4 had a closerelationship with 16S rRNA gene of Serrratia marcescens ATCC 13880 (similarity: 99.53-99.8%), Eschericia coliBA 30.1 with Eschericia coli ATCC 11775T (similarity: 99.38-99.67%), Salmonella typhi BA 07.4, Salmonella typhiKD 30.4, and Salmonella typhi SA 02.2 with Salmonella typhi ATCC 19430T (similarity: 99.4-100%) as well as theisolates of Enterobacter cloacae SA 02.1, Enterobacter cloacae BA 45.4.1, one 16S rRNA gene clone of Enterobactercloacae TG 03.5 with Enterobacter cloacae ATCC 23373 (similarity: 99.0-99.87%).
Chemosystematic of Enterobacteriaceae Familia Obtained from Blood Cultures Based on Total Protein Profiles Darmawati, Sri; Sembiring, Langkah; Asmara, Widya; Artama, Wayan T.; Anwar, Syaiful
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (303.344 KB) | DOI: 10.22146/ijbiotech.7862

Abstract

The purpose of this study was to determine the chemosystematic of 14 strains of bacteria in blood cultures from Semarang using 1 reference strain S. typhi NCTC 786, based on the total protein profi les with the similarity relationship analysis based on Simple Matching Coeffi cient (SSM) analysis and algorithm methodof unweighted pair group with averages (UPGMA) presented in a dendrogram. The results showed that thechemosystematic based on the total protein profi les using SDS-PAGE method can classify the member ofbacterial strains of each species. The Clusters respectively consist of 4 strains of S. typhi (similarity: 89.7%),2 strains of Ser. marcescens (similarity: 89.7%), two strains of E. coli, and one strain of Salmonella ssp, S. typhi NCTC 786 (similarity: 100%). Those three incorporated clusters had the similarity value of 75.3%. Those four strains of Ent. cloacae composed in one cluster (similarity: 100%) are incorporated in a cluster consisting of one strain of Kleb. pneumoniae (similarity: 92.9%). Both clusters were incorporated in a cluster consisting of S. typhi NCTC 786 (similarity: 67.9%). Key words: Enterobacteriaceae, chemosystematic, blood cultures, protein profile