I Made Artika

Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody. Key words: allotropic expression, ATP synthase, mitochondria, yeast

Laboratory biosafety for handling emerging viruses

Abstract Emerging viruses are viruses whose occurrence has risen within the past twenty years, or whose presence is likely to increase in the near future. Diseases caused by emerging viruses are a major threat to global public health. In spite of greater awareness of safety and containment procedures, the handling of pathogenic viruses remains a likely source of infection, and mortality, among laboratory workers. There is a steady increase in both the number of laboratories and scientist handling emerging viruses for diagnostics and research. The potential for harm associated to work with these infectious agents can be minimized through the application of sound biosafety concepts and practices. The main factors to the prevention of laboratory-acquired infection are well-trained personnel who are knowledgable and biohazard aware, who are perceptive of the various ways of transmission, and who are professional in safe laboratory practice management. In addition, we should emphasize that appropriate facilities, practices and procedures are to be used by the laboratory workers for the handling of emerging viruses in a safe and secure manner. This review is aimed at providing researchers and laboratory personnel with basic biosafety principles to protect themselves from exposure to emerging viruses while working in the laboratory. This paper focuses on what emerging viruses are, why emerging viruses can cause laboratory-acquired infection, how to assess the risk of working with emerging viruses, and how laboratory-acquired infection can be prevented. Control measures used in the laboratory designed as such that they protect workers from emerging viruses and safeguard the public through the safe disposal of infectious wastes are also addressed.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus

BackgroundInfluenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009.ResultsAn optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested.ConclusionsThe developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical ‘ladder-like’ phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.

Biosorption Copper (Cu) and Mercury (Hg) by Omphalina sp. using Batch, Rotary, Biotray, and Pack Bed Flow Methods

The agricultural potential in Indonesia still becomes the excellence because Indonesia is the agrarian country. The role of agricultural sector in the national economy is empirically proven to be real enough both in quite normal economy and when the economy facing the crisis. This is seen from two important indicators i.e. the contribution to the sector of Gross Domestic Product (GDP), and the labor absorption that always dominates in the agricultural field.This is caused by the lack of competence of farmer to be able to develop in the agricultural sector. With this is needed the farmer competence in forming the farming capacity building so the farmer can play the role in the community and can compete in the current globalization era. Based on the above problem, the research aims to: (1) analyze how the competence is owned by the farmer in Sumatera, (2) obtain the picture of farmer superior competence model in Sumatera to form the capacity building of highland vegetable farmer, and (3) give any solution which should be done in order to be able to increase the farmer competence in Sumatera. In this research, for analyzing the farmer superior competence and the making of superior competence model in forming the capacity building of highland vegetable farming in Karo District, it is used the ISM, AHP, and IPA.Ethanol is considered as the most promising alternative fuel, since it can be produced from a variety of agriculturally-based renewable materials, such as sugarcane bagasse. Lignocellulose as a major component of sugarcane bagasse is considered as an attractive renewable resource for ethanol production due to its great availability and relatively low cost. The major problem of lignocellulose is caused by its need for treatment to be hydrolyzed to simple sugar before being used for bioethanol production. However, pretreatment using acid as hydrolyzing agent creates some inhibitor compounds that reduce ethanol production because these compounds are potential fermentation inhibitors and affect the growth rate of the yeast. Reduction of these by-products requires a conditioning (detoxification and culture starter adaptation). Thus, the aim of this study was to evaluate bioethanol production by fermentation with and without detoxified sugarcane bagasse acid hydrolysate using adapted and non-adapted culture of C. tropicalis. According to this study, the highest ethanol amount was obtained about 0.43 % (v/v) with an ethanol yield of 2.51 % and theoretical yield of 4.92 % by fermentation of sugarcane bagasse hydrolysate with detoxification using the adapted strain of C. tropicalis at 72 hours fermentation time. Furthermore, the addition of 3 % glucose as co-substrate on detoxified-hydrolysate media only achieved the highest ethanol concentration 0.21 % after 24 hours fermentation with the ethanol yield 0.69 % and theoretical ethanol yield 1.35 %, thus it can be concluded that the addition of glucose could not increase the ethanol production.Cassava leaves (Manihot esculenta Crantz) widely used as vegetables in many regions. Cassava leaves contain many minerals such as Fe, Zn, Mn, Cu, Mg, Ca, P, K, S, contain crude protein, β-carotene and have an active compound of flavonoids, phenolic, and contain chlorophyll which is a natural antioxidant. The aims of this study were to analyze the effect of boiled cassava leaves on total phenolics, flavonoids, and its antioxidant activity. Cassava leaves extraction was done by maceration method to obtain the methanol extract and infundation method to get the water extract. Based on phytochemical test, cassava leaves contained alkaloids, flavonoids, phenolic, tannins, and saponins. The highest levels of total phenolics and flavonoids contained in the methanol extract of simplicia, 30.57 mg GAE/g and 881.33 mg RE/g, respectively. Cassava leaves extract had potential as an antioxidant with the highest inhibition of DPPH radicals produced by the methanol extract of simplicia with IC 50 values of 92.10 mg/L and followed by methanol extract of boiled leaves, water extract of simplicia, and water extract of boiled leaves with IC 50 144.28, 155.76 dan 170.71 mg/L , respectively. In conclusion, the process of boiling cassava leaves could reduce total phenolic, total flavonoids and antioxidant activcity.

Amplification and Analysis of Cytocrome Oxidase I of Polypedates leucomystax from Bogor Agricultural University Area

DNA barcoding has become a useful tool for identifying and confirming of species within a known taxonomic framework. A large-scale effort is underway to barcode all amphibian species using the universally sequenced DNA region, a partial fragment of mitochondrial cytochrome oxidase subunit I (COI). This study was aimed to use DNA barcoding technique to identify and confirm species of Polypedates leucomystax and to analyze their phylogenetic relationship. Samples of Polypedates leucomystax were collected from Campus Area of Bogor Agricultural University. The cytochrome oxidase I gene of 600-700 nucleotides were amplified and observed in agarose gel electrophoresis. Forward sequence (604 base pairs) of COI gene was used for phylogenetic analyses. BLAST analysis against BOLD System database showed 95.75% identity with sequences of Polypedates leucomystax. The pairwise genetic distances of Polypedates leucomystax with Rhacophorus schlegelii, Limnonectes fujianensis, Fejervarya cancrivora, and Bufo melanostictus were 0.274, 0.352, 0.339, 0.339, 0.393, respectively. These results illustrated that the genetic identification is congruence with the morphological identification. Phylogenetic tree analysis showed that the samples were in one clade with other tree frogs. The DNA barcoding technique based on the sequence of COI gene can therefore be used to identify and confirm species of Polypedates leucomystax.

Isolation and Molecular Cloning of Cellulase Gene from Bovine Rumen Bacteria

Cellulases are the enzymes that hydrolyze cellulosic biomass and are produced by the microorganisms that grow over cellulosic matters. The objective of this research was to isolate and clone cellulase gene from cellulose-degrading bacteria of bovine rumen. Cellulose-degrading bacteria was isolated from rumen fluid using a selective medium. Total RNA was isolated from selected colony having cellulose degrading activity and was used as a template for cDNA construction using reverse transcriptase polymerase chain reaction (RT-PCR) technique. The resulted cDNA was employed as a template for PCR amplification of cellulase gene using specific primers. The cellulase gene candidate obtained was cloned into the pGEM-T-Easy vector followed by determination of its nucleotide sequence. The sequence was then aligned with sequences of cellulase genes from GenBank. Results showed that a number of isolates of rumen bacteria exhibit cellulase activity and the CR-8 isolate was selected for further analysis. The successful isolation of total RNA from CR-8 was indicated by the presence of two intense bands of ribosomal RNA (23S and 16S). The reverse transcription process was successful and the amplification of cellulase gene using the specific primers F1 and R1 resulted in a DNA fragment of 1900 bp as a candidate of cellulase gene. The fragment was successfully cloned into the pGEM-T-Easy vector, and the resulted recombinant plasmid was successfully introduced into the E. coli cells. Nucleotide sequence analysis suggested that the cloned gene is cellulase gene and shares 99% homology with the endo-1,6-beta-glucanase of T. harzianum.

Phylogenetic Analysis of Cytochrome Oxidase I from Buduk Toads Duttaphrynus melanostictus and Phrynoidis asper from Bogor

Indonesia have high diversity of Amphibians. Amphibians have an important role in ecosystem and produce many bioactive peptides. However, the genetic information of amphibians from Indonesia is very limited, especially Duttaphrynus melanostictus and Phrynoidis asper. The aims of this study are to determine the nucleotide sequence of cytochrome oxidase I (COI) from D. melanostictus and P. asper, to analyze their genetic diversity and their phylogenetic relationship. A total 668 base pairs of COI gene fragment were successfully amplified and their nucleotide sequence determined. P. asper (5 haplotypes) samples group have high haplotype diversity compared to D. melanostictus (1 haplotype). The results of Basic Local Alignment Search Tools (BLAST) to the NCBI and BOLD database, showed 99 % - 100 % identity to sequence of D. melanostictus. For the sequence of P. asper showed 99.23 % identity to sequence P. asper in BOLD database. There was no sequence of COI gene of P. asper in NCBI database. Genetic relationship among species in family Bufonidae, indicated that D. melanostictus has closer relation to P. asper than to another species, inspite of their pharapyletic characteristic. For intern species relationship of D. melanostictus, the data showed that D. melanostictus from Bogor have closer relationship to D. melanostictus from India than D. melanostictus from China.

Activity of Skin Secretions of Frog Fejervarya limnocharis and Limnonectes macrodon against Streptococcus pneumoniae Multidrug Resistant and Molecular Analysis of Species F. limnocharis

Indonesia has about 450 frog species which is approximately 20% of frog species in the world. Among frog species found in Indonesia are Fejervarya limnocharis dan Limnonectes macrodon belonging to family Dicroglossidae. Frog skin secretion is considered to have a potency to be used as an alternative source of antibacterial agent against Streptococcus pneumoniae multidrug resistant (MDR). The aims of the present study were to analyze antibacterial activity of skin secretions of F. limnocharis and L. macrodon against S. pneumoniae multidrug resistant (MDR) and conduct molecular phylogenetic analysis of the frog used to ensure classification of frog species. The release of skin secretion was stimulated using epinephrine injection. Antibacterial activity of the skin secretions was tested using the well and paper disc methods. Results showed that skin secretions of F. limnocharis have antibacterial activity against S. pneumoniae multidrug resistant (MDR) SPN1307. The activity, however, was lower compared to that of chloramphenicol in both well and paper disc methods. On the other hand, skin secretions of L. macrodon failed to inhibit the growth of S. pneumoniae multidrug resistant (MDR) SPN1307. Molecular phylogenetic analysis was carried out on F. limnocharis based on DNA sequence of a partial fragment of mitochondrial cytochrome oxidase subunit I (COI) gene. Results showed that the frog F. limnocharis is closely related (97%) to Fejervarya sp from Bali. Skin secretions of F. limnocharis, therefore, has the potency to be developed as a source of antibacterial agents against S. pneumoniae multidrug resistant (MDR) SPN1307.

The Activity of Wungu Leaf ( Graptophyllum pictum (L) Griff) Extract in Reducing Blood Glucose Level of Hyperglycemic Mice

Wungu leaf (Graptophyllum pictum (L.) Griff) is a plant thought to have potential use in alleviating symptoms of diabetes mellitus. The purpose of the present study was to evaluate the activity of wungu leaf extracts in decreasing blood glucose level of alloxan (200 mg/kg BW)-induced hyperglycemic mice. Extracts of wungu leaf were obtained by macerating with ethanol and then partitioning the extract with diethyl ether, ethyl acetate, and butanol. Each extract obtained was used to treat hyperglycemic mice for 28 days. The results showed that wungu leaf extracts have the ability to decrease the blood glucose level of hyperglycemic mice (dose 50 mg/kg BW). The ethyl acetate extract showed the highest activity, bringing about a decrease of blood glucose of 37.6 %. The wungu leaf extract has the potential to be developed as a source of anti-diabetic agents.