DIAH ISKANDRIATI
Primate Research Center Bogor Agricultural University, Bogor, Indonesia

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Expression of Simian Retrovirus Type D Serotype 2 Envelope in Insect Cell Using Baculovirus Expression Vector System ISKANDRIATI, DIAH; SADIKIN, MOHAMAD; PAMUNGKAS, JOKO
Microbiology Indonesia Vol 3, No 2 (2009): August 2009
Publisher : Indonesian Society for microbiology

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Abstract

Simian retrovirus type-D (SRV) is a causative agent of simian acquired immunodeficiency syndrome in Asian macaques, and can serve as a viral model in understanding of retrovirus infection because of some similarities to human AIDS pathogenesis. Study of infection and pathogenesis of SRV in macaques could be a strategy of vaccine and antiviral development for preventive and therapeutic purposes. We expressed the SRV-2 envelope gene using baculovirus expression vector system and transfected it to Spodoptera frugiferda insect cell line for SRV-2 recombinant protein production. Analysis using PCR and sequencing technique of recombinant in the passage-3 viral stock indicated the occurrence of recombination between SRV-2 envelope and baculovirus genome. Purification using immobilized metal ion affinity chromatography Ni2+-NTA to recombinant protein could minimize the presence non-specific proteins. The SDS-PAGE analysis showed a specific protein for SRV-2 gp70 envelope. Western blot analysis of this purified protein indicated a specific reaction with anti-SRV-2 antibody positive of Macaca fascicularis serum shown as SRV-2 gp70 envelope band.
Isolation and Characterization of Simian Retrovirus Type D from Macaca fascicularis and M. nemestrina in Indonesia ISKANDRIATI, DIAH; SAEPULOH, UUS; MARIYA, SILMI; GRANT, RICHARD F; SOLIHIN, DEDY DURYADI; SAJUTHI, DONDIN; PAMUNGKAS, JOKO
Microbiology Indonesia Vol 4, No 3 (2010): December 2010
Publisher : Indonesian Society for microbiology

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Simian type D retroviruses (SRVs) are one of the causative agents of simian acquired immunodeficiency syndrome (AIDS) in Asian macaques.  In the past, SRV isolates from macaques had only been identified at the US primate centers, outside the country of origin and after the animals had been introduced into a new environment.  In this study, we report the first isolation, cultivation and molecular characterization of the type D simian retrovirus naturally infecting wild caught macaques in their natural habitats in the country of origin, in this case, Indonesia.  When peripheral blood mononuclear cells (PBMC) from Macaca fascicularis (Mf) and M. nemestrina (Mn) were co-cultured with Raji human B-cell line, syncytia were observed microscopically and confirmed by immunofluoresence assay using antibody to SRV-2.  Immunoblot analysis of purified Mf-ET1006 from cell culture supernatants demonstrated that the viral core and envelope proteins reacted with rabbit anti-SRV.  Sequence analysis of Mf isolates in the viral envelope region revealed high homology to SRV-2 (94-96%). On the other hand, the homologies in the envelope region of Mn isolates were less than 80% to SRV-1, SRV-2, SRV-3 and Mf isolates.   This study suggests that the isolate from Mn may be different from any other published SRV isolates.
Dissemination in Pigtailed Macaques after Primary Infection of Dengue-3 Virus PAMUNGKAS, JOKO; ISKANDRIATI, DIAH; SAEPULOH, UUS; AFFANDI, MOSES; ARIFIN, ESTHER; PARAMASTRI, YASMINA; DEWI, FITRIYA NUR ANISA; SAJUTHI, DONDIN
Microbiology Indonesia Vol 5, No 2 (2011): June 2011
Publisher : Indonesian Society for microbiology

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Nonhuman primates (NHPs) play as indispensable animal model in biomedical research for studying a variety of human health issues, diseases and disorders, therapies, and preventive strategies. Since the immunological and physiological responses of NHPs, at some extent, to experimental viral infections are similar to humans, it is possible that studies of dengue infection in NHPs may aid understanding of dengue infection in humans. In this study,we used pigtailed macaques (Macaca nemestrina) as the experimental animal to study dengue-3 (DEN-3) virus infection.We evaluated DEN-3 viral distribution and replication sites after a primary infection in all collected tissues. Sequential localization in tissue of DEN-3 virus was studied in pigtailed macaques euthanized three days post viral inoculation (10 pfu mL ). Pigtailed macaque that was inoculated subcutaneously or intravenously; showed the highest viremia (62.94 pfu mL and 58.62 pfu mL ) detected by one step reverse transcription real time PCR. The virus inoculated in pigtailed macaques by subcutaneous injection was rapidly disseminated from the inoculation site to the lymph nodes, adrenal glands, kidneys, heart, thyroid, liver, prostate gland, and seminal vesicles. Meanwhile, dissemination of dengue virus in pigtailed macaques inoculated intravenously was detected in lymph nodes, thymus, salivary glands, liver, and prostate gland. This study suggested that the above mentioned-tissue specimens are involved or affected by DEN-3 virus replication and the route of infection seemed to have influenced the virus dissemination.  
Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli SAEPULOH, UUS; ISKANDRIATI, DIAH; HOETAMA, FUNGKEY; MARIYA, SELA SEPTIMA; SOLIHIN, DEDY DURYADI; PAMUNGKAS, JOKO; SAJUTHI, DONDIN
Microbiology Indonesia Vol 7, No 2 (2013): June 2013
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.7.2.3

Abstract

In this study, we isolated the simian betaretrovirus serotype-2 (SRV-2) reverse transcriptase (RT) gene from infected Indonesian cynomolgus monkey (Macaca fascicularis). The gene was then cloned in Escherichia coli expression system. The SRV-2 RT gene is located between nucleotides 3284-4925 in the polyprotein (Pol) region encodes 547 amino acids. Analysis of expression using SDS-PAGE and western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 U μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of β-globin and β-actin genes produced DNA fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.
Biocompatibility of various hydoxyapatite scaffolds evaluated by proliferation of rat’s bone marrow mesenchymal stem cells: an in vitro study Kamal, Achmad F.; Iskandriati, Diah; Dilogo, Ismail H.; Siregar, Nurjati C.; Hutagalung, Errol U.; Susworo, R.; Yusuf, Achmad A.; Bachtiar, Adang
Medical Journal of Indonesia Vol 22, No 4 (2013): November
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (516.004 KB) | DOI: 10.13181/mji.v22i4.600

Abstract

Background: Scaffold (biomaterial) biocompatibility test should be performed in vitro prior to in vivo stem cell application in animal or clinical trial. These test consists of direct and indirect toxicity test (MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). Those tests were used to identify cell morphological changes, cell-substrate adhesion impairment, and reduction in cell proliferation activity.Methods: The tested scaffolds were hydroxyapatite-calcium sulphate (HA-CaSO4) (scaffold I), nano-particular HA paste (scaffold II), synthetic HA granule (scaffold III), bovine HA granule (scaffold IV), and morsellized bovine xenograft (scaffold V). Direct contact toxicity test and MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were performed on those groups. In direct contact toxicity test, we put granules of various scaffolds within plates and incubated together with mesenchymal stem cells (MSCs). In MTT assay we included phenol 20 mg/mL and 100 mg/mL group as positive control. Morphology, cell adhesion impairment, and cell growth were monitored daily until day-7. Cells counting in the direct contact toxicity test was conducted on day-7.Results: There were no changes on 24 hours observation after direct contact. On day-7, an impairment of cell adhesion to plastic substrates, changes in cell morphology, and cell death were observed, especially in scaffold I, scaffold II, and scaffold V. In MTT assay, only scaffold I, phenol 20 mg/mL, and phenol 100 mg/mL showed more than 50% inhibition at 24-hour and 7-day-observation. Extracts from scaffold II, III, IV, and V did not affect the viability and proliferation of bone marrow MSCs (inhibition value < 50%). Scaffold II, III, IV and V were proven non-cytotoxic and have good biocompatibility in vitro,  no statistical significant differences were observed among the scaffold groups (p > 0.05).Conclusion: We understand which scaffold was nontoxic or the least toxic to MSCs in vitro. Scaffold IV (bovine HA granule) showed the least toxic effect to rat’s bone marrow MSCs on direct contact test and MTT assay. (Med J Indones. 2013;22:202-8. doi: 10.13181/mji.v22i4.600)Keywords: Biocompatibility test, direct contact test, hydroxyapatite, MTT assay, scaffold
Ekspresi Enzim Rekombinan Reverse Transcriptase (RTRNase H) Simian Betaretrovirus Serotipe-2 Asal Macaca fascicularis Indonesia dalam Sistem Ekspresi Eschericia coli Saepuloh, Uus; Iskandriati, Diah; Pamungkas, Joko; Sajuthi, Dondin
Jurnal Ilmu Pertanian Indonesia Vol 18, No 1 (2013): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

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Abstract

Reverse transcriptase (RT) enzyme is a key tool in molecular biology for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). Combining RT activity with PCR amplification has been a gold standard as the first step in cloning the coding region of any gene of interest. Evidently, RTs have been critical in advancing molecular biology, genetics and medicine to their current stage. In this study, we were developing the RTDRNase H recombinant enzyme isolated from serotype-2 simian betaretrovirus-2 (SRV-2) infected Indonesian Macaca fascicularis using Escherichia coli expression system. The study was conducted using RT SRV-2 gene expression using E. coli expression system, proteins purification, and application to RT PCR technique. The SDS PAGE expression analysis showed a specific band size of 32.7 kDa assumed as RT protein enzyme. Application of RT SRV-2 enzyme generated to the RT PCR technique of β-globin CDV and SRV-2 env gene target showed that the RT SRV-2 enzyme was capable to reverse transcribed mRNA into cDNA as indicated by the presence of specific DNA band compared with commercial RT enzymes. This RT SRV-2 enzyme showed its activity similar to that of commercial one, although the activity was lower. Keywords: escherichia coli expression system, recombinant enzyme, reverse transcriptase, SRV-2
Analysis of Intestinal Mucosal Immunoglobulin A in Sprague Dawley Rats Supplemented with Tempeh SOKA, SUSAN; SUWANTO, ANTONIUS; RUSMANA, IMAN; SAJUTHI, DONDIN; ISKANDRIATI, DIAH; JESSICA, KATHARINA
HAYATI Journal of Biosciences Vol 22, No 1 (2015): January 2015
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1507.883 KB) | DOI: 10.4308/hjb.22.1.48

Abstract

Tempeh is a well-known Indonesian fermented food made from soybean. During the fermentation process, microorganisms play an important role in the flavor, texture, and nutritional quality of tempeh. Tempeh has been show to have immuno-modulatory and immune-stimulating properties that may also be caused by the microorganisms in tempeh as they interact between the microbial population in the intestinal tract. The objective of this study was to quantify IgA gene expression at both the transcription and translation levels in Sprague Dawley (SD) rats supplemented with tempeh. A total of 6 female SD rats were divided into 3 groups of 2 rats. The first group was the control and was fed a standard diet without tempeh. The second- and third group were fed with a standard diet supplemented with raw and cooked tempeh, respectively. Ileum tissue samples were collected after tempeh supplementation for 28 days. RNA was extracted from ileum samples, and measurement of IgA gene expression was further analyzed using semi quantitative real-time PCR. The concentration of IgA protein was quantified from ileum lysate using the half sandwich ELISA method. IgA gene expressions in rats supplemented with raw, and with cooked tempeh, were 1.18 and 1.17 fold higher, respectively, compared to the control group. Moreover, IgA protein secretion levels also increased 2.46 and 2.08 fold, respectively, compared to the control group. The result of this study indicates that both raw and cooked tempeh may stimulate IgA secretion, and also that both viable and non-viable microorganisms might stimulate IgA gene expression.
Ekspresi Enzim Rekombinan Reverse Transcriptase (RTRNase H) Simian Betaretrovirus Serotipe-2 Asal Macaca fascicularis Indonesia dalam Sistem Ekspresi Eschericia coli Saepuloh, Uus; Iskandriati, Diah; Pamungkas, Joko; Sajuthi, Dondin
Jurnal Ilmu Pertanian Indonesia Vol 18, No 1 (2013): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (295.513 KB)

Abstract

Reverse transcriptase (RT) enzyme is a key tool in molecular biology for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). Combining RT activity with PCR amplification has been a gold standard as the first step in cloning the coding region of any gene of interest. Evidently, RTs have been critical in advancing molecular biology, genetics and medicine to their current stage. In this study, we were developing the RTDRNase H recombinant enzyme isolated from serotype-2 simian betaretrovirus-2 (SRV-2) infected Indonesian Macaca fascicularis using Escherichia coli expression system. The study was conducted using RT SRV-2 gene expression using E. coli expression system, proteins purification, and application to RT PCR technique. The SDS PAGE expression analysis showed a specific band size of 32.7 kDa assumed as RT protein enzyme. Application of RT SRV-2 enzyme generated to the RT PCR technique of β-globin CDV and SRV-2 env gene target showed that the RT SRV-2 enzyme was capable to reverse transcribed mRNA into cDNA as indicated by the presence of specific DNA band compared with commercial RT enzymes. This RT SRV-2 enzyme showed its activity similar to that of commercial one, although the activity was lower.
Identifikasi Molekuler Virus Papilloma Genital Pada Dua Spesies Primata di Fasilitas Penangkaran Pusat Studi Satwa Primata-Institut Pertanian Bogor Sari, Isti Kartika; Suparto, Irma H; Iskandriati, Diah
JURNAL BIOLOGI INDONESIA Vol 10, No 1 (2014): JURNAL BIOLOGI INDONESIA
Publisher : Perhimpunan Biologi Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (144.5 KB) | DOI: 10.14203/jbi.v10i1.339

Abstract

Tulisan Pendek
Mammary Gland Cell Culture of Macaca fascicularis as a Reservoir for Stem Cells Mariya, Silmi; Dewi, Fitriya Nur Annisa; Suparto, Irma Herawati; Wilkerson, Gregory K.; Cline, J. Mark; Permanawati, .; Iskandriati, Diah; Budiarsa, I Nengah; Sajuthi, Dondin
HAYATI Journal of Biosciences Vol 24, No 3 (2017): July 2017
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1422.555 KB) | DOI: 10.4308/hjb.24.3.136

Abstract

The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys (Macaca fascicularis)(MfMC) and furthermore identified the expression of markers for stemness and estrogenreceptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24, CK18, and marker for fibroblast S1004A. Importantly, the cell population also consistently expressed the markers of mammary stem cells (ITGB1 or CD29 and ITGA6 or CD49f), mesenchymal stem cells (CD73 and CD105) and pluripotency (NANOG, OCT4, SOX2). In addition to this, the cells were also positive for Estrogen Receptor (ER), and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future.