2 research outputs found

    Overexpression of host furin protease and inhibitory activities of synthetic chalcones- and azepines- derivative compounds toward dengue virus type-2 / Khuzaidatul Azidah Ahmad Nazri

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    The outbreak of dengue disease continues to occur despite extensive measures of vector control. Meanwhile, progress in the vaccine development is often affected by various challenges thus delaying in the production of an effective vaccine across all four dengue serotypes. Thus, efforts in combating dengue are being channelled to other alternatives such as dengue antivirals. The search for this therapeutics has given rise to various screening methods to test for potential inhibitory activities of purified as well as synthetic compounds. Therefore, one of the aims of this study is to produce a fiirin recombinant protein. This current study also aims to determine the inhibitory effect of the synthetic chalcones- and azepines-derivatives toward dengue virus infection in vitro. To achieve the first objective, the furin gene isolated from HepG2 cells inoculated with dengue virus type-2 (DENV2) was cloned and overexpressed in the E.coli. The protein lysates of the overexpressed protein were purified using Ni²⁺ (resin) affinity chromatography and its concentration was measured by Bradford assay. The purified furin was confirmed by SDS-PAGE and Western blot analysis. The result showed that the furin is expressed at 60 kDa and was positive toward Rabbit Monoclonal Anti-Furin antibody. Subsequently, two groups of synthetic chalcones (2446DA and 20H46DA) and azepines (MA13, MA15 and MA16) derivatives were measured for their inhibitory activities toward dengue infection by cytotoxicity assay, plaque assay, indirect immunostaining, in vitro inhibition assay and fluorescence scanning microscopy. The cytotoxicity assay results showed that the concentration below maximum non-toxic dose (MNTD) for both 2446DA and 20H46DA in HepG2 cells was 15 μg/mL. The same value was obtained for MA15 and MA16. However, MA13 was observed to be less toxic compared to all test compounds with MNTD of 30 μg/mL. The plaque forming unit per ml (pfu/m1) was reduced prominently by 10 to 1000 fold when the infected BHK21 cells were treated with the highest non-toxic concentration compared to lowest non-toxic concentration. The indirect immunostaining results showed a similar trend of virus particles reduction on infected HepG2 cells in both the chalcones- and azepines-derivatives when treated at simultaneous- and post- conditions. However, the azepines MA13 exhibited the most potent activity towards DENV2 whereby total inhibition of virus particles was observed during simultaneous-treatment condition. The in vitro inhibition assay results showed that at concentration below MNTD, all compounds exhibited inhibitory activity against DENV2 in a dose dependent manner, indicated by the absence of cytopathic effects. The inhibitory potency strength exhibited was between 73% to 100% against both 1000 TCID₅₀ (p>0.05) and 100 TCID₅₀ (p<0.05). Results of the fluorescence scanning microscopy showed that all cytoskeletal changes induced by DENV2 infection were managed by the inhibitory activity of chalconesand azepines-derivatives in BHK21 cells. In conclusion, we proposed that the purified furin host protease to have high substrate specificity and highly potential to be developed as screening tool for anti-furin compounds. More importantly, the synthetic chalcones- and azepines-derivatives are suitable candidates for the development of therapeutics against dengue infection

    Palm tocotrienol-rich fraction protects neonatal rat cardiomyocytes from H2 O2 - induced oxidative damage

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    Oxidative stress plays an important role in the pathogenesis of heart disease. Tocotrienol-rich fraction (TRF) is an antioxidant and that has the potential to reduce the risk of heart disease. This study is to determine the protective effects of palm TRF against H2 O2 -induced oxidative damage in neonatal rat cardiomyocytes (NRCM). The NRCM were divided into control, treated with TRF (10 µg/mL), H2 O2 (0.5 mM) and treated with TRF prior to H2 O2 induction (pre-treatment). Cell viability was determined by the MTS assay,while the presence of reactive oxygen species (ROS) was determined using fluorescent dihydroethidium (DHE) and 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA) dye. Mitochondrial integrity and cell death were determined using JC-1 and Annexin V-FITC staining, respectively. Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were determined by colorimetric assay kit. The concentration of H2 O2 from 0.5 to 5 mM reduced the cell viability and the H2 O2 IC50 value of 0.5 mM was used in the experiment. H2 O2 induction increased the intensity of carboxy-H2 DCFDA and DHE-stains; and also the intensity of green fluorescence of J-monomers in JC-1 staining compared to the control group. The activity of LDH increased while the activity of SOD decreased in the H2 O2 group. Pre-treatment with TRF reduced the intensities of carboxy-H2 DCFDA and DHE-stains, as well as the green fluorescence of J-monomers in JC-1. Meanwhile, the LDH activity was reduced in the pre-treatment group but no changes were recorded in SOD activity compared to the H2 O2 group. Palm TRF protects cardiomyocytes from oxidative damage by reducing ROS production and maintaining the mitochondrial membrane integrity thus reducing cell death
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