Indonesian Journal of Biotechnology
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A Single Base Substitution Adjacent to the Stop Codon in the downstream of the SMP3 gene Affects its Post-trancriptional process in Saccharomyces cerevisiae
No full textThe smp3-1 mutant allele confers increased holding stability of heterologous plasmid, pSR1, and a temperature-sensitive growth defect which is remediable by the addition of 1 M sorbitol as the osmotic stabilizer. The smp3-1 allele contains two base substitutions; one is in the open reading frame and changed the 490th CAT (encoding Histidine) to TAT (tyrosine), and the other one is an A for G substitution, at 2 bp downstream from termination codon. These base substitutions were separated each other by recombination at a BstNI site located between these two substitutions. The base substitution in the 3'' untranslated region was found to be lethal and the defect was unremediable by the osmotic stabilizer, while that in the open reading frame has no appreciable effect to the cell. Thus, both the base substitutions join together confer the smp3-1 mutant phenotype. The smp3-1 mutant cells cultivated at 37 OC in nutrient medium containing 1 M sorbitol showed similar smp3 transcription as in the wild type. These facts suggest that smp3-1 mutation has a defect in its post-transcriptional process
Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer
No full textMarine actinomycetes is a robust source of secondary metabolites including anticancer compounds . The objective of this research was to select marine actinomycetes producing potential compounds as anticancer used combination methods that consist of amplification PKS I (polyketide synthases type I) and NRPS (non ribosomal peptide synthetases) genes, analysis the diversity of secondary metabolites and genetic. Selected isolates were used for cytotoxicity assay. PKS I and NRPS genes were amplified using sets of degenerate primers. K1F and M6R were used for amplify ketosynthase and methyl-malonyl-CoA transferase modules of PKS I gene which targeted sequences 1200-1400 bp. A3F and A7R were used for amplify adenilation domains of NRPS gene which targeted sequences 700-800 bp. The diversity of secondary metabolites was analized by TLC and densitometry of ethyl acetate extracts. Genetic diversity was analized by repetitive DNA fingerprinting using BOXA1R primers. The cytotoxicity of secondary metabolites on T47D and MCF7 breast cell lines cancer was measured by MTT assay method. Fifty two marine actinomycetes isolates were screened using combination methods. Ten isolates were detected encoding both PKS I and NRPS genes, whereas 11 isolates were detected encoding the NRPS gene. The screening by analysis of secondary metabolites and genetic diversity methods were obtained 6 selected isolates for cytotoxicity assay, which consist of 3 isolates encoding both PKS I and NRPS genes and 3 isolates encoding NRPS gene.Isolate 1 had high cytotoxicity with the IC50 on T47D cell was 19 μg/ml and the IC50 on MCF7 cell was 7 g/ml. This findings suggests that combination methods were effective and efficient way to select marine actinomycetes producing potential compounds as anticancer
T47D cells arrested at G2M and Hyperploidy Formation Induced by a Curcumin’s Analogue PGV-1
No full textits chemical structure than curcumin. As a curcumin analogue, PGV-1 was considered to have anticanceractivities. This research was conducted to study the effect of PGV-1 on the cycle progression of T47D cells. Cytotoxiceffects of PGV-1 on T47D cells were determined using MTT assay, and the the effect on cell cycle progressionwas carried out using flowcytometry. Western blot analysis was used to analyze protein expression correspondingto cell cycle progression. The result showed that at the concentration of 2.5 μM PGV-1 inhibited cell cycleprogression through G2/M arrest and induced of cells hyperploidy formation. The hyperploidy formation inducedby PGV-1 was related to the increase of cdc-2 expression. PGV-1 2.5 μM elevated the level of p21 CIP/KIPthrough p53- independent manner. Apoptosis was also induced by PGV-1 at early phase of treatment indicated byPARP cleavage due to activation of caspase-3/7 after 12 h treatment. The results above suggest that PGV-1 inhibitsthe growth of T47D cells targeted on microtubules.Keywords: PGV-1, G2/M arrest, apoptosis, p2
Reactive Oxygen Intermediate (ROI) in Dog Macrophage Infected with Mycobacterium tuberculosis
No full textexperiment used 24 healthy dogs, aged between 1 and 2 years, both male and female which were divided into twodifferent groups consisting of 12 dogs each. The first group was the treatment group, that is they were infected with Mtuberculosis and the second one was the control group. The activity of macrophages ROI secretion were measured at1st, 2nd, 12th, and 24th after infection using nitroblue tetrazolium (NBT) reduction assay. Three cats were used to measure themacrophage activity in each period, using triplicate sample for each cat. The results of the experiment showed thatROI secretion increased in infected group compared with the control group, and this activity reached to the plateaulevel at 2 weeks after infection. Although these enhanced activities were gradually diminished thereafter, higherlevels of these activities were consistently observed until the end of experiment compared with control group. Theresults of the experiment indicated that ROI played an important role to against M.tuberculosis infection in dogs.Keyword: macrophage, ROI, M.tuberculosis, dog
In vitro Antiplasmodial Activity and Cytotoxicity of Vincadifformine and Its Semisynthetic Derivatives
No full textAn indole alkaloid with aspidospemane structure possessing a potential antiplasmodial activity, vincadifformine, has been isolated from Aspidosperma pyrifolium Mart. Moreover, 10 derivatives were prepared from the vincadifformine. The study was conducted to evaluate the in vitro antiplasmodial and cytotoxic activity of the vincadifformine and their semisynthetic derivatives. The in vitro antiplasmodial activity was evaluated on Plasmodium falciparum chloroquine-resistant (FcM29) and –sensitive (Nigerian) strains after 24-h and 72-h incubation, while cytotoxic activity was estimated on Hela cells and Cytotoxicity Index (CI = IC50 on HeLa cells/IC50 on FcM29 strain) was calculated to evaluate the safety of tested compounds. Experiment results showed that two compounds (4 and 8) exhibited good antiplasmodial activities in comparison with parent compound, vincadifformine and other tested compounds with IC50 ranging from 5.3 to 12.8 µM on FcM29 strain and 11.4 to 24.0 µM on Nigerian strain. In addition, the CI of two compounds were also lower after 24-h incubation (CI, 2.0 and 4.8) than that of after 72-h incubation (CI, 9.5 and 11.5). Further study will be conducted to evaluate quantitative structure-activity relationship (QSAR) in order to design new antimalarial drugs
Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR
No full textworld. 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 PC
Production of Poly-α-hydroxybutyrate (PHB) from Sago Starch by The Native Isolate Bacillus megaterium PSA10
No full textA new bacterial strain that produces amylase and poly-α-hidroxybutyrate (PHB) using sago starch as carbon source was characterized and identified to be member of the Bacillus megaterium group based on phenotypic characteristics and 16S rDNA gene sequences. Amylase activity was determined spectrophotometrically on the basis of substrate concentration reduction. PHB production was quantified with UV spectrophotometer. The polymer produced by B. megaterium PSA10 was identified by Fourier Transform Infrared spectroscopy (FTIR). The result of the study showed that the amylase specific activity B. megaterium PSA10 was 593,61 DUN/mg protein and PHB production from sago starch was 52,28 % of cell dry weight (CDW). FTIR analysis of the polymer indicated that the strain B.megaterium PSA10 was a potent PHB producer
Detection of eae, bfpA, espA Genes on Diarrhoeagenic Strains of Escherichia coli Isolates
No full textThe Enteropathogenic Escherichia coli (EPEC) is one of pathogenic strain of diarrheagenic E. coli group in children and infant that occurs in developing countries. The significant virulence factors in pathogenic EPEC are eaeA (E. coli attaching- effacing ), bfpA (bundle-forming pilus A) and espA (encoding secreted protein A) genes. The use of DNA probes to detect the virulence genes in E. coli in Indonesia is not common yet. In this experiment the gene fragments of eae, bfpA, and espA were used as probes to detect the EPEC among E. coli isolates from stool specimensin of diarrheic children attending Public Health Centers in Yogyakarta. The DNA samples were isolated from 49 diarrheagenic E. coli isolates. The DNA probes of eae, bfpA and espA were obtained by amplification of DNA fragment of EPEC O126 using PCR technique. Furthermore, those probes were used to identify the presence of those genes among E. coli isolates using hybridization technique. The results showed that 42 (85.7%) isolates were espA+, 25 isolates (51%) were eaeA+ (EPEC strains). Therefore among 25 isolates of EPEC, 20 isolates (80 %) among EPEC were bfpA+ (typical EPEC strains)
The Efficacy of Fucoidan on Gastric Ulcer
No full textHyperacidity causes gastric injury, and in severe situations, ulcer could develop. The growth factors known asthe basic fibroblast growth factor (bFGF) and the epidermal growth factor (EGF) have been recognized to promoteulcer healing. Fucoidan is extracted from a brown seaweed of Okinawa called Mozuku or Cladosiphon okamuranus.Fucoidan is effective for the healing of gastric ulcers by inducing epithelial cells to produce growth factors. The aimof this study is to explore the efficacy of fucoidan in patient who suffered by gastric ulcer. A randomized control trialdouble blind was conducted to 33 eligible samples. By using four-blocks random samples were divided into fucoidanand placebo groups. 100 mg of fucoidan was given to the fucoidan group and 100 mg of glucose was given to theplacebo group. Due to ethical reasons, for both groups were given a proton pump inhibitor. There was no differencein the age category between the fucoidan group (mean: 46.23 ± 14.8 years) and the placebo group (mean: 46.18 ± 18.4years) (p: 0.28). There was also no difference in sex between the fucoidan group (female: 10/33; male 7/33) and theplacebo group (female: 7/33; male: 9/33); p: 0.38. According to the SAKITA and MIWA criterias 32 patients fulfilledA1 which indicate active severe ulcer, and 1 patient fulfilled A2 which indicate active moderate ulcer. Most of theulcers were gastric ulcer. There was a significant improvement of the grade of ulcer in fucoidan group (94%) (16/17)compared to placebo group (37.5%) (6/16,p: 0.005). There was a significant reduction of abdominal pain after 5 daysin the fucoidan group, compared to the placebo group (p: 0.04). Vomiting tends to decrease in day 6 of the fucoidangroup however its proportion is similar with that of the placebo group (p: 0.9). Fucoidan is effective for ulcer healingand reducing ulcer symptoms.Key words : fucoidan, gastric ulcer, anti-peptic activit
Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates
No full textMycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis. Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulation of immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate within macrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis by INH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level were measured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, Roche Diagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, Scientific Device Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tb clinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates was shown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no association between macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulent Mtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparable level