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

The Use of HIS6 Gene as a Selectable Marker for Yeast Vector

The yeast Saccharomyces cerevisiae HIS6 gene has been shown to be functional as a selectable marker for selecting and maintaining a yeast vector in yeast S. cerevisiae host cells. The yeast HIS6 gene encodes an enzyme involved in the yeast histidine biosynthesis. The yeast HIS6 gene was cloned into a yeast expression vector. The resultant recombinant plasmid was introduced into yeast host cells defective in endogenous HIS6 gene. The functionality of the HIS6 gene as a selectable marker was tested by growing transformed cells on selective minimum medium lacking histidine supplementation. Key words: HIS6 gene, selectable marker, vector, transformation, yeas

HISTONE ACETYLTRANSFERASE P300/CBP-ASSOCIATED FACTOR INHIBITION BY QUERCETIN AS ANTICANCER DRUG CANDIDATE WITH IN SILICO AND IN VITRO APPROACH

Objective: The objective of this research was to show quercetin potency to inhibit histone acetyltransferase p300/CBP-associated factor (HAT PCAF) activity. Molecular docking study was used to show inhibition model of quercetin towards HAT PCAF and the kinetic study was used to give the information about inhibition constant (Ki) of quercetin.Methods: Molecular docking simulations between HAT and quercetin were performed using AutoDock Vina, and the results were scored based on its Gibbs free energy change (ΔG) (the most negative ΔG). The kinetic assay of HAT PCAF inhibition by quercetin used fluorometry methods to measure enzyme inhibition by quercetin. Results: Molecular docking showed that quercetin could inhibit HAT PCAF through binding to acetyl-CoA that involved glutamine 525 (Gln525) and cysteine 574 (Cys574) on chain A, and Cys574 and Gln581 on chain B of HAT PCAF. Quercetin also binds to histone active site on HAT PCAF through aspartic acid 610 (Asp610). The kinetic study results showed that quercetin could inhibit histone acetylation based on the fluorescence intensity. Analysis by Dixon plot showed that quercetin competes with histone. Therefore, ithad competitive inhibition. Its Ki value of 9.575 µM. Kinetic study showed the same result as molecular docking study that quercetin had potency as an HAT PCAF inhibitor.Conclusion: The result of this research showed that quercetin had the potency to inhibit HAT PCAF through competition with HAT PCAF substrates. Quercetin could interact with the HAT PCAF active site, thus, lower the HAT PCAF activity. Keywords: Histone acetyltransferase, Quercetin, PCAF, Epigenetic drug, Dockin

Bioenergetic Consequences of FLAG Tag Addition to the C-Terminus of Subunit 8 of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase

The yeast mitochondrial F1F0-ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. FLAG tag addition to subunit 8 protein potentially facilitate elucidation of its topology, structure, and function. It has been shown that following incorporation of FLAG tag to its C-terminus, subunit 8 still assemble into functional ATP synthase complex. In order to analyze bioenergetic consequences of the FLAG tag addition, a yeast strain expressing FLAG tagged-subunit 8 was subjected to cellular respiration assays. Results obtained showed that addition of FLAG tag to the C-terminus of subunit 8 does not impair its proper functioning. The FLAG tag system, therefore, can be employed to study subunit 8’s detailed structure, topology, and function

Sosialisasi Tanggap Darurat Pencegahan Covid-19 di Kepulauan Talaud

Since the World Health Organization or WHO declared that the Coronavirus disease (Covid-19) is a pandemic, the President of the Republic of Indonesia and the Regional Government and their staff have worked hand in hand to make some tactical steps to prevent the spread of this virus. The public must be informed on the dangers of Covid-19, through creation of special protocols which are expected to prevent the spread of this virus. The purpose of this activity was to conduct socialization directly or indirectly in providing education, convincing, and providing an overview of the dangers of the Covid-19 Virus and applying health protocols to the community in Melonguane District, Talaud Islands Regency, North Sulawesi Province. This activity took place from July 7 to August 28, 2020. The results of this activity showed that the people in the Talaud Islands have understood the importance of using good and correct masks when outside the home and also the use of hand soap or hand sanitizer which can help people to avoid this dangerous virus. Keywords: covid-19, emergency response, Kepulauan Talaud, Melonguan

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, yeas

Biosorpsi Tembaga (Cu) dan Merkuri (Hg) oleh Omphalina SP Menggunakan Metode Batch, Rotary, Biotray, dan Pack Bed Flow

Penelitian ini bertujuan untuk menentukan metode yang paling efektif pada biosorpsi logam oleh Omphalina. Biomassa yang masih hidup dapat menyerap logam lebih banyak dari pada biomassa yang sudah mati, sehingga perlu dilakukan optimalisasi penggunaan biomasa dengan membandingkan metode batch, Rotary, packbed flow, biotray. Penelitian dilakukan pada larutan Cu 100 dan 200 ppm, dan Hg 3 dan 5 ppm, dengan variasi waktu kontak 1, 3, 5, 7, dan 24 jam , masing-masing sampel diukur kandungan logamnya dengan menggunakan spektrofotometri serapan atom. Hasil penelitian menunjukkan bahwa Hg dengan konsentrasi awal 3 ppm dapat berkurang hingga 91.38 % pada metode Rotary, 83.98% biotray, 87.14% pack bed flow, 32.94% batch, sedangkan pada Cu dengan konsentrasi awal 100 ppm dapat berkurang hingga 23.58% pada metode Rotary, 22.66%. biotray, 10.53% pack bed flow, dan 10.17% pada batch. Penyerapan optimal Hg dan Cu terjadi pada 1 jam pertama dan kapasitas serapan logam lebih tinggi pada metode rotary

The characterization of bacteriocins produced by Lactobacillus plantarum strains isolated from traditional fermented foods in Indonesia and the detection of its plantaricin-encoding genes

Lactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents

Antibacterial Activity of Leaf Extracts of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. Against Streptococcus pneumoniae

Antibacterial resistance in Streptococcus pneumoniae has been increasing and is one of ongoing global concern.  The need to find new antibacterial agents against Streptococcus pneumoniae is of paramount importance. Medicinal plants are prospective sources of antibacterial agents. The aims of the present study were to determine the activity of leaf extraxt of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. against Streptococcus pneumoniae.  Leaves of Anredera cordifolia (Ten.) Steenis were extracted using 96% ethanol, while the leaves of Muntingia calabura L were extracted using 100% methanol.  The leaf extracts of the two plants obtained were bioassayed for antibacterial activity against Streptococcus pneumoniae ATCC 49619 and a clinical isolate Streptococcus pneumoniae PU 067.  Results showed that leaf extracts of both Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. have antibacterial activity in vitro against Streptococcus pneumoniae ATCC 49619 at crude extract concentrations of 25%, 50%, 75% and 100% (w/v). Both plants extracts showed strongest activity against S. pneumoniae ATCC 49619 at extract concentration of 75%.   In addition, the extracts of both plants have inhibitory activity against growth of the clinical isolate Streptococcus pneumoniae PU 067. Both plant extracts showed strongest activity against S. pneumoniae PU 067 at extract concentration of 100%.  Therefore, leaf extracts of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. can potentially be used as a source of antibacterial agent for Streptococcus pneumoniae. Keywords: Antibacterial agent, Anredera cordifolia (Ten.) Steenis, Muntingia calabura L., Streptococcus pneumoniae

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