Construction and eukaryotic expression of recombinant large hepatitis delta antigen

Background:

Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging.

Methods:

In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages.

Results:

Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy.

Conclusion:

Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research.Key Words: Hepatitis Delta Virus, L-HDAg, SOEing-PCR     

Ethanol and value‐added byproducts from rice straw by dimorphic fungus Mucor hiemalis

Different morphologies of Mucor hiemalis were induced and used for the production of ethanol and biomass from rice straw through a separate hydrolysis and fermentation process. The yield of enzymatic hydrolysis was improved from 40.4% for the untreated straw to 80–93% by employing sodium hydroxide and concentrated phosphoric acid pretreatments with or without ultrasonication. The best hydrolysis performance was achieved after pretreatment by sodium hydroxide assisted with ultrasonication. The ethanol yields from the hydrolysates were 0.39–0.44 g/g depending on the pretreatment method and the fungus morphology. The yeast‐like form of the fungus showed faster glucose assimilation and slightly higher ethanol yield compared to the other morphologies. The biomass yield of mostly yeast‐like cells was more than the other morphologies (0.202–0.282 g/g glucose). Moreover, the biomass of the yeast‐like cells had more protein content (46.7–52.4 %) compared to filamentous cells (37.7–46.3 %). The cell wall, alkali‐insoluble material (AIM) of the biomass, represented 16.3–20.1% of the biomass. On average, total chitin‐chitosan content of AIM of the biomass of purely filamentous, mostly filamentous, mostly yeast‐like, and purely yeast‐like forms of the fungus was 0.460, 0.373, 0.330, and 0.336 g/g AIM of the biomass, respectively.     

Design and synthesis of antimicrobial,anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone

We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition reactions. Structures of these compounds were established on the basis of ¹H NMR, ¹³C NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial, anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules, some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to have good activity.     

Effects of silicon oxide nanoparticles on growth and physiology of wheat seedlings

The effects of silicon oxide (SiO₂) nanoparticles at concentrations of 50, 100, 200, 400, and 800 mg/L on Triticum aestivum L. seedlings were investigated. We showed that SiO₂ nanoparticles, at concentrations higher 200 mg/L, had negative impacts on wheat seedlings. At these concentrations, SiO₂ nanoparticles significantly decreased roots and shoots fresh weight, decreased roots and shoots dry weight, decreased amounts of chlorophyll a and b in leaves, decreased amount of carotenoids in leaves, increased proline content in leaves, increased lipid peroxidation in leaves, and increased catalase activity in leaves. Results of this study indicate that at lower concentrations (such as 50 and 100 mg/L), SiO₂ nanoparticles not only have negative effects on wheat seedlings, but can have even some positive effects.     

Caryophyllene Sesquiterpenes from Pulicaria vulgaris Gaertn.: Isolation,Structure Determination,Bioactivity and Structure−Activity Relationship

A new caryophyllene, named pulicaryenne A ( 1 ), along with four other known caryophyllene derivatives ( 2 , 3 , 4 and 5 ) were isolated from the ethyl acetate extract of aerial parts of Pulicaria vulgaris Gaertn . (Asteraceae). All compounds were isolated for the first time from this species. Compound 2 was identified as a new epimer of a known caryophyllene derivative isolated previously from P. dysenterica. Their structures were established by spectroscopic means including NMR analysis (1D‐ and 2D‐NMR) and ESI‐TOF‐MS. All compounds were evaluated for their anticholinesterase, antityrosinase and cytotoxic activities against two human cell lines (A549 and HeLa). Results showed that compound 5 exhibited the highest cytotoxic effect against A549 and anticholinesterase activity with IC₅₀ values of 8.50±0.75 and 6.45±0.09 μg/mL, respectively. Furthermore, compound 5 showed also an interesting antityrosinase activity with percent inhibition value of 79.0±2.5 % at 50 μg/mL. The bioactivity and drug likeness scores of the isolated compounds 1–5 were calculated using Molinspiration software and discussed. These results may suggest that the five caryophyllene derivatives endowed with good biological properties, which could be used as bioactive alternatives in pharmaceutical preparations.     

A simple innovative spectrofluorometric method for the determination of alendronate in bulk and in pharmaceutical tablets

Sodium alendronate is the first in a pharmacological class known as bisphosphonates, used for treatment of various bone diseases. Assay of bisphosphonates by a spectroscopic technique is very challenging due to the fact that they lack chromophores and none of them are fluorescent. In this work, a simple method is presented for determination of alendronate in bulk and in pharmaceutical tablets using spectrofluorometry by exploiting the ability of alendronate to displace salicylate from the iron(III)–salicylate chelate, forming a non‐fluorescent colorless iron(III)–alendronate complex. The liberated salicylate is fluorescent and is equivalent to the mount of alendronate added. The response was linear over the concentration range 20–90 μM and the proposed method was validated according to the guidelines of the International Conference on Harmonization. The correlation coefficient was found to be 0.995 and the limit of detection was 7.5 μM. The method was successfully applied for determination of alendronate in the commercially available Osteonate® tablets. The average percent recovery ± percent relative standard deviation was found to be 102.118 ± 2.033 which is congruent with the label claim of the dosage form. The results were also compared to a reported method using t‐test and F‐test at 95% confidence level; no significant differences were observed. The presented method is simple, fast, easy, cost‐effective and suitable for routine pharmaceutical analysis.     

Cucurbitacins from Ecballium elaterium juice increase the binding of bilirubin and ibuprofen to albumin in human plasma

Ecballium elaterium, a medicinal plant, whose fruit juice is used for the treatment of jaundice in folk medicine, has been reported as being capable of decreasing bilirubinemia in animals with jaundice [H.H. Elayan, M.N. Garaibeh, S.M. Zmeili, S.A. Salhab, Effects of Ecballium elaterium juice on serum bilirubin concentration in male rats, Int. J. Crude Drug Res. 27 (1989) 227-234]. The aim of this study is to identify the Ecballium elaterium components, which are able to modify the binding of bilirubin to albumin. The juice is fiber-free but contains proteins, lipids, sugars, and minerals. The extract of the juice, analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), contains cucurbitacins (Cuc) B, D, E, and I as well as several glycosylated compounds. Human plasma containing no or serial concentrations of Ecballium elaterium components were prepared and the direct bilirubin (DB) and total bilirubin (TB) were determined by the Jendrassik and Grof method. Our results showed that Cuc D, E, and B decreased the levels of DB and TB in plasma, while Cuc I, glycosyl derivatives, and proteins of the juice did not modify the bilirubin levels. The binding of domain specific ligands to HSA, bilirubin (domain IIA), and ibuprofen (domain IIIA), were studied in the absence and presence of Cuc D, E, and I, by fluorescence spectroscopy. The values of binding constant K(a) and binding site number n, determined by Scatchard method, increased for the both ligands only in the presence of Cuc E and D. Cuc I decreased slightly the K(a) of ibuprofen, suggesting an interaction with the domain IIIA of the protein. As a conclusion, Cuc E, D, and B produce rearrangement in the structure of albumin leading to increase the binding of domain specific ligands, ibuprofen and bilirubin.