Annales Bogorienses

12-17

Authors: Sri Hartati, Marissa Angelina, Lia Meilawati, Indah Dwiatmi Dewijanti

The flavone glycoside was isolated from ethyl acetate fraction of ethanol extract of leaves Pterocarpus indicus Willd. The isolation conducted by gravitation column chromatography and eluted successively with hexane, ethyl acetate and methanol by gradient, further and purified by sephadex-LH20. The structure was elucidating base on spectroscopy data of NMR proton and carbon one and two dimension, UV, LC-MS and FT-IR. Antioxidant was evaluated using 2,2-diphenyl-1-picrylhidrazyl radical scavenging. The isolation and identification led a stigmasterol (1) and a new flavonol-glycoside or ptevon-3-D- glycoside (2), and antioxidant activity are showed IC50 18.53 m mol for compound 2 and blank of quercetin 7.94 m mol and Vitamin C 40.25 m mol. These compounds and antioxidant activities are the first time reported from this plant.

23-28

Authors: Nina Artanti, Faiza Maryani, Hani Mulyani, Rizna Triana Dewi, Vienna Saraswati, Tutik Murniasih

The marine bacteria were cultured in liquid medium under shaking condition were extracted with ethyl acetate. Antidiabetes was measured using inhibition of α-glucosidase inhibitory activity method; antioxidant was measured using DPPH free radical scavenging activity method; antibacterial was tested using disc diffusion method.S creening results showed that at sample concentration of 200 µg/ml, there was significant α-glucosidase inhibitory activity was detected in the extracts of strain sp 7.9 (84% inhibition) and 8.10 (75% inhibition),however the antioxidant activity of these two strains were low only around 30% inhibition, antioxidant activities of other strains were very low.Screening for antibacterial activities using 10µl samples showed that extract of strain Sp 8.5was best for Staphylococcusaureus (14 mm inhibition); Sp 7.9 and Sp 8.5 for Bacillus subtilis (18 mm inhibition); Sp 8.10 for Escherichia coli (10 mm inhibition); Sp 8.9 and Sp 8.10 for Pseudomonas aeuriginosa. Based on these results marine bacteria strain Sp 7.9 and Sp 8.10 were selected to be used for further studies in the isolation of bioactive that has potential as antidiabetes and antibacterial.Results of molecular identification conducted by INACC showed that identity of both strain based on BLAST Homology using NCBI database were Bacillus thuringiensis strain Ou2.

9-16

Authors: Endah Puji Septisetyani, Arizah Kusumawati, Adi Santoso

Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies may depend upon the type or amount of cationic lipids, the cell line or DNA plasmid being used for transfection. The purpose of this study was to find optimal condition for transfection of CHO-K1 and CHO-S cells with pJ-EPO plasmid (contain human epo gene) compared with pEGFP-c1 plasmid (contain gfp gene) by cationic lipid Lipofectamin 2000TM to generate stable transfectant expressing recombinant human erythropoietin (rhEPO). Optimization was carried out regarding the amount of lipofectamin, DNA concentration, and concentration of antibiotic Geneticin (G418) for selection of stable transfectants. Using standard amount of lipofectamin (10µl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 4µg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, and 10µg/well pJ-EPO plasmids. The data also indicated that optimal transfection conditions of CHO-K1 and CHO-S cells with pJ-EPO were shown with the use of 4µg/well DNA and 15µl lipofectamin, respectively. Concentration of G418 affected the expression where strongest rhEPO expression was shown at 1,000 ng/µl G418 concentration.

29-36

Authors: Dyah Retno Wulandari, Agus Purwito, Slamet Susanto, Ali Husni, Tri Muji Ermayanti

Tetraploid citrus are important for interploidal hybridization to create triploid seedless citrus. Colchicine is the most commonly used as antimitotic agent to induce polyploid plants. Tetraploid induction by colchicine in Pummelo ‘Nambangan’ was conducted in vitro using different types of explants. The aim of this research was to induce tetraploid pummelo ‘Nambangan’ by colchicine treatment using germinated seed, shoot tip and cotyledonary node as explants. Tetraploid shoot induction was conducted by soaking germinated seeds, shoot tips and cotyledonary nodes in 0.1% colchicine for 1, 3 and 5 h. Regenerant shoots were grown on MS medium and their growth was observed after four weeks in culture. Ploidy level was determined using flow cytometry analysis. Stomata density, length and width of stomatal guard cell were also recorded. The results showed that shoot elongation was inhibited by colchicine treatment. Soaking of shoot tip explants in 0.1% colchicine for 1 h resulted in 66.66% of putative tetraploid shoots. Compared to diploid shoots, tetraploids had lower stomata density but bigger in guard cell size.

13-25

Authors: Ari Fina Bintarti, Iman Rusmana, Aris Tri Wahyudi

Metanotrophic bacteria have ability to oxidize methane and fix atmospheric nitrogen, hence the bacteria has an important role as a nitrogen source provider on wetland area like rice fields. Nitrogen fixation process is catalyzed by the nitrogenase enzyme complex, encoded by nifD and nifH genes. However, characteristic of these genes from indigenous-methanotrophic bacteria still poorly understood. Hence, nifD and nifH genes of methanotrophic bacteria isolated from rice fields in Indonesia (BGM3, BGM9, SS1, SS3, SS10, ST18, SP3 and INP4) were identified and characterized. Detection of nifH and nifD genes was conducted by polymerase chain reaction (PCR) amplification. nifH and nifD gene sequences were analyzed using BLAST-X and phylogenetic trees were constructed using Neighbour Joining method. Based on nifH sequences analysis, SS1 closely related to Beijerinckia mobilis and SS3, SS10, ST 18 closely related to Beijerinckia indica subsp. indica ATCC 9039, while, BGM3, INP4, and BGM9 related to nifH of uncultured nitrogen-fixing bacterium. In other hand, sequence analysis of nifD gene showed that SS1, SS3, SS10, ST 18 closely related to B. indica subsp. indica ATCC 9039 and BGM3, BGM9, INP4 closely related to Xanthobacter autotrophicus Py2. Identification by 16 SrRNA indicated that SS1, SS3, SS10, and ST18 had closeness to Beijerinckia sp. P310-1, while INP4 closely related to Xanthobacter sp. M5C24.

35-44

Authors: Wien Kusharyoto, Ira Handayani, Martha Sari, Asrul Muhamad Fuad

An ideal therapeutic for cancer would be one that selectively targets to tumor cells, is nontoxic to normal cells, and that could be systemically delivered, thereby reaching metastases as well as primary tumor. Immunoliposomes directed by monoclonal antibody or its fragments are promising vehicles for tumor targeted drug delivery. Transferrin receptors (TfR) levels are elevated in various types of cancer cells and considered to correlate with the aggressive or proliferative ability of tumor cells. Therefore, TfR levels can be elaborated as a prognostic tumor marker, and TfR is a potential target for drug delivery in the therapy of malignant cells. Here, we report the preparation of an anti-TfR single-chain antibody variable (scFv) immunoliposome for tumortargeted delivery vehicle. The cDNA encoding the variable heavy and light chain domains of the anti-TfRscFv antibody fragment was derived from the murine monoclonal antibody Clone E6, which is specific towards transferrin receptor. The gene encoding the anti-TfR scFv fragment was codon optimized for expression in Escherichia coli, subsequently synthesized, and cloned into the expression vector pJexpress404. The His6- tagged anti-TfR scFv fragment was expressed in E. coli and purified by means of immobilized metal-ion affinity chromatography on TALON™ matrix. SDS-PAGE revealed that the scFv fragment had the size of approximately 27 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence. Liposome containing 5% MPB-DOPE were prepared by ethanol injection method. Afterwards, the anti-TfR scFv fragments were covalently conjugated to the liposome to produce the anti-TfR scFv immunoliposome with the size of around 200 to 300 nm.

25-34

Authors: Hariyatun Hariyatun, Antonius Suwanto, Wien Kusharyoto

Intimin is the main adhesin of Enterohemorrhagic E. coli (EHEC) O157:H7 bacteria which are the most common leading infectious cause of bloody diarrhea and acute kidney failure in children who develop hemolytic uremic syndrome (HUS). Intimin is required for persistent bacterial colonization to eukaryotic host cell and its receptor-binding activity is localized at the C-terminus 282 amino acids (Intimin282). Thus, Intimin282 is an attractive antigen candidate that could be useful in vaccine and diagnostic systems against EHEC infections. Previous studies had reported expression of Intimin in E. coli cytoplasm using commonly used prokaryotic expression systems. However, it usually encountered several problems, i.e. low expression level, leaky expression, inclusion body formation, and truncated protein. The pRHA vector, which is tightly regulated by Lrhamnose and D-glucose, represents a viable alternative E. coli expression system to overcome such problems. Moreover, E. coli periplasm has an advantage of maintaining protein functionality by providing an oxidative environment that is more efficient than cytoplasm. However, to date there is no study about Intimin expression using pRHA expression system and/or in E. coli periplasm. Accordingly, we constructed a recombinant pRHA vector harbouring the respective gene to investigate the expression of an immunogenic Intimin fragment of EHEC O157:H7 in E. coli periplasm. The gene encoding His6-tagged Intimin282 (Int282) together with pelB signal sequence was cloned into the pRHA vector, subsequently expressed in E. coli JM109 and purified. Expression and purification of Int282 were verified by SDS-PAGE and Western blot. The result showed that Int282 was successfully expressed in E. coli periplasm with a protein size of approximately 32 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence.

45-53

Authors: Tri Muji Ermayanti, Erwin Al Hafiizh, Ary Mandessy, Gesang Setyadi, Andi Mukhsia

Exploitation on plant population may put the endemic plants into an endangered state, hence, these plants will need to be conserved. In order to pursue conservation on endemic plants, we conducted in vitro seed germination and shoot multiplication of seven alpine and sub-alpine species endemic to Mount (Mt.) Jaya, in Papua, Indonesia, i.e. Tetramolopium klossii, Deschampsia klossii, Papuacalia cartenszensis, Epilobium hooglandii, Gaultheria novoguinensis, Rhododendron correoides and Rhododendron culminicolum. These species are categorized as slow-growth plants found in higher altitude (over 3700 m above sea level) and low temperature of Mt. Jaya. Seeds were surface-sterilized using Na-hypochloride and germinated aseptically on Murashige and Skoog (MS) medium. Dytikinin benzyl adenine (BA) was used for shoot multiplication. Seedling cultures were maintained in a controlled environment with continuous low light intensity (800 lux) and at temperature 26-27oC. Results showed that most species had more than 80% of germination rate on MS medium after a week in culture. BA was required to enhance shoots multiplication. Woody Plant (WP) (Lloyd & McCown, 1981) medium gave better shoot multiplication for R. culminicolum.