Indonesian Journal of Biotechnology
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Comparison of Cytotoxic and Antiproliferative Effects of Benzylidenecyclopentanone Analogues of Curcumin on RBL-2H3 Cells
No full textCurcumin is a natural yellow pigment isolated from the rhizomes of Curcuma longa L. (turmeric), and has several pharmacological effects and no toxicity in both in animal and human clinical study. However, the problem of curcumin is its stability because of its active methylene moiety. Modification of this moiety to cyclopentanone is expected to increase the stability. Previous study reported that benzylidenecyclopentanone analogues of curcumin showed inhibitory effect on histamine release from RBL-2H3 (rat basophilic leukemia) cells, a tumor analog of mast cells. One of them, the hydroxy-methoxy analog (PGV-0), showed more potent effect than that of curcumin. In the present study, some benzylidenecyclopentanone analogues of curcumin were evaluated for their effects on the viability and proliferation of RBL-2H3 cells. Viable cells were counted under a light microscope with a cells-counting chamber or using the cell viability reagent WST-1. The results showed that mast cell viability and histamine content were not affected by curcumin and benzylidene cyclopentanone for 30 min incubation, however, impaired for overnight incubation. The hydroxy-dimethyl benzylidene analog (PGV-1) strongly decreased the mast cells viability for overnight incubation, and its effect was highest among the other analogues. In the proliferation study, this compound also strongly inhibited the proliferation of mast cells, whereas curcumin and hydroxy-methoxy benzylidene analog inhibited the proliferation slightly. There were no inhibitory effects on mast cells proliferation treated by dibenzylidene; dihydroxybenzylidene; and hydroxy-diethylbenzylidene cyclopentanone.Keywords : viability, proliferation, curcumin, benzylidene cyclopentanone, RBL-2H3 cell
Ethanol Production by Fermentation of Various Sweet-Stalk Sorghum Juices Using Various Yeast Strains
No full textThe ethanol production by fermentation of sweet-stalk sorghum juice is affected by the juice composition and the capability of the yeast strain to ferment it. Eight yeast strains were tested on their growth and ethanol fermentation abilities in sweet-stalk sorghum juices extracted from three cultivars of sweet sorghum. The best specific growth rate of the yeast strains grown aerobically in the yeast extract peptone dextrose (YEPD) broth and the sweet-stalk sorghum juices of KCS105, FS501, and FS902 cultivars, were achieved by OUT7903, OUT7913, OUT7903, and OUT7027 yeast strains, respectively. However, the best specific CO2 evolution rate of the yeast strain during fermentation of the juices was achieved by OUT7027 yeast strains. The highest ethanol concentration, ethanol yield, and sugar conversion efficiency (SCE) were obtained by strain OUT7921 when it was employed to ferment sweet-stem sorghum juice of FS902 cultivar. It was also observed that the juice extracted from sweet-stalk sorghum of FS902 cultivar is the most suitable medium for all yeast strains to achieve their best fermentation abilities. Thus, it is likely that the growth and ethanol production ability of a yeast strain in sweet-stalk sorghum juice depend on the physiological responses of the yeasts to nutrientcomposition of the sorghum juice and the sorghum cultivar from which the juice was extracted.Key words : Sweet-stalk sorghum juice, ethanol, fermentation, yeas
Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java
No full textThermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR). Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, clonin
Detection and Cloning of a Gene Involved in Zwitermicin A Synthesis from Plant Growth Promoting Rhizobacteria of Bacillus sp CR64
No full textUtilization of soil bacteria as biocontrol agent is becoming popular due to its valuable and effective mechanisms to suppress plant pathogenic microbes. We have previously isolated Bacillus sp, designated as Bacillus sp CR64, which exhibited effective plant growth promoting and antifungal activities. In this study, CR64 was examined in inhibiting the growth of Rhizoctonia solani, the causing agent of root rot disease. Partial sequence analysis of 16S rRNA gene revealed that this isolate similar with Bacillus cereus (94%). Furthermore, a gene designated zmaR was detected by means of specific amplification of DNA fragment approximately 950 bp. This fragment was then cloned onto pCRII-TOPO (3.9 kb) and sequenced using DNA sequencer ABI PRISM 310. Sequence analysis revealed that it had highest homology with the ZmaR protein (89% identity; 90% similarity) of B. thuringiensis serovar kurstaki (AAF82729.2). Alignment analysis with other ZmaR sequences from other antibiotic-producing Bacilli exhibited an almost fully conserved region within ZmaR sequences.Key words : PGPR, Bacillus sp CR64, Zwitermicin A, Cloning, Antifungal
Effect of Nuclear Export Inhibitor Leptomycin B on the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells
No full textLeptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of manyregulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants.In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cel
An Actinomycetes Producing Anticandida Isolated from Cajuput Rhizosphere: Partial Identification of Isolates and Amplification of pks-I genes
No full textActinomycetes have been the most prolific producer of various kinds of antifungal metabolites, and many of them are described as being produced by polyketide synthetases (pks). We present strain of Actinomycetes producing anticandida isolated from rhizosphere plant for amplification of Pks-I genes. The isolate was obtained from Wanagama I Forest UGM Yogyakarta. Gene of seven isolates, from total of 173 isolates, were amplified using degenerate primer to detect the presence of pks genes. One strain that is named Streptomyces sp. GMR-22 was partialy identified as anticandida producing actinomycete. The strain shown the strongest activity against Candida albicans. Based on bioautography assay, one spot active with Rf 0.57 was appeared as bright yellow by cerrium sulphate but it was and not visible on UV254 and 366 lights. Key words : pks genes, anticandida, Streptomyces sp GMR-22, rep-PCR, cajuput rhizospher
The Distribution of MAP-2 Phosphorylation in Cerebral Cortex of Long-Tailed Monkey Fetuses (Macaca fascicularis) in the Last Trimester of Gestation
No full textMemories are storage in cholinoceptive cells, the cells which are enriched with microtubule-associated protein 2 (MAP-2) that localized in the neuronal dendrite and the cell bodies. Phosphorylation of MAP-2 may increase memory with reduce stability of dendrite by altered dendrite length and lead new side-branches of neuronal as a neuronal plasticity processes in cerebral cortex. The aim of this research is to study the distribution of MAP-2 phosphorylation neurons in cerebral cortex of long-tailed macaques in the third semester of gestationalimmunohistochemically using avidin biotin conjugated complex method. Neurons MAP-2 phosphorylation immunoreactive were located in dendrites and cell bodies, mostly in pyramidal neurons of cerebral cortex. Intensity of MAP-2 phosphorylation immunoreactivity in layer V were stronger than another layer and the neurons that very intensely stained were the pyramidal cells in frontal and parietal lobes, that was suggested that neurons in this areas more responsive to neuroplasticity. From the results we concluded that MAP-2 phosphorylation already distributed in the cerebral cortex of long-tailed macaque fetuses at the last trimester of gestation, mostly in the pyramidal cells of layer V that is suggested plays a role for preparation of memoryformation.Keywords: fetus, long-tailed monkey, cerebral cortex, memory, MAP-2 phosphorylatio
Ethanolic Extract of Hedyotis corymbosa L. Increases Cytotoxic Activity of Doxorubicin on MCF-7 Breast Cancer Cell
No full textHedyotis corymbosa L. with ursolic acid as the main compound is one of the plants that has been used for traditional medicine including to cure breast cancer disease. The aim of this research is to examine the cytotoxic activity of rumput mutiara herb ethanolic extract (ERM) and its effect in combination with doxorubicin against MCF-7 breast cancer cell line as cell model of doxorubicin resistance. Hedyotis corymbosa L. herb powder extraction was done by maceration using ethanol 96% then the extract is detected for ursolic acid content. Cell viability assay of ERM, doxorubicin and the combination of ERM and doxorubicin treatments were carried out by MTT assay to determine IC50 and CI (Combination Index). Cell cycle distribution was determined by flowcytometry. Apoptosis assay was performed by ethidum bromide-acridine orange DNA staining method. Investigation on Bcl-2 expression was determined by immunocytochemistry method. Thin Layer Chromatography of ERM had similar Rf with ursolic acid standard: 0,6. ERM and doxorubicin inhibited cell growth against MCF-7 with IC50 of 77 µg/mL and 349 nM (0,19 µg/mL) respectively. Combination of ERM and doxorubicin showed synergistic effect (CI 0.66-0.99). Combination of 25 ìg/mL ERM- 200 nM doxorubicin induced apoptosis and decreased Bcl-2 expression but showed no cell accumulation on cell cycle. Doxorubicin induced high cell accumulation in G2/M phase, but ERM at the concentration of 25 ìg/mL had a low effect in G1 phase, and ERM IC50 did not induce cell accumulation otherwise apoptosis. These results concluded that the apoptosis mechanism of combination doxorubicin-ERM is mediated by cell cycle arrest and non cell cycle arrest. Therefore ERM has a potential activity to be developed as co-chemotherapeutic agent.
Decolorization of Remazol Briliant Blue R by Laccase from White Rot Fungus Polyporus sp. S133
No full textThe decolourization of the recalcitrant dye RBBR by the culture filtrate of Polyporus sp. S133 and its isolatedlaccase was investigated. The laccase alone decolorized RBBR. A small molecular weight redox mediator (HBT) wasnecessary to increase the decolorization. The purified laccase totally decolorized the dye of 200 mg l-1 initialconcentration of RBBR when only 1.5 U ml-1 of laccase was used in the reaction mixture. The effects of differentphysicochemical parameters were tested and optimal decolorization rates occurred at pH 5 and at a temperature of 50°C. The effect of surfactants on the decolourization of RBBR was tested with Tween 80, Tween 20, and Brij 35. It wasdemonstrated that Tween 80 was inhibiting substrate for the decolorization while Tween 80 and Brij 35 was noinhibiting effect for the decolorization. Provided that all of the condition is included, it is suggested that laccase maybe suitable for the wastewater treatment of similar anthraquinone dyes.Keywords: Decolorization; Laccase; Remazol Brilliant Blue R (RBBR); Polyporus sp. S13
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-a-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