Correlation between total phenolic and flavonoid contents and biological activities of 12 ethanolic extracts of Iranian propolis.

Propolis is a resinous substance produced by honey bees that is very popular as a natural remedy in traditional medicine. The current research is the first study on the biological properties of ethanolic extracts of propolis (EEP) from several different regions (12) of Iran. Total phenolic and flavonoid contents (TPC and TFC) of Iranian EEPs were variable between 26.59-221.38 mg GAE/g EEP and 4.8-100.03 mg QE/g EEP. The DPPH scavenging assay showed all the studied EEP samples, except for the sample with the lowest TPC and TFC (P6), have suitable antioxidant activity. All the EEPs inhibited both cholinesterase enzymes (acetylcholinesterase: AChE, butyrylcholinesterase: BuChE) but most of them exhibited a distinct selectivity over BuChE. Evaluation of the antibacterial activity of the EEP samples using four pathogenic bacteria (B. cereus, S. aureus, A. baumannii, and P. aeruginosa) demonstrated that the antibacterial properties of propolis are more effective on the gram-positive bacterium. Spearman correlation analysis showed a strong positive correlation between TPC and TFC of the Iranian EEPs and their antioxidant, anticholinesterase, and antibacterial activities. Considering that there is ample evidence of anticholinesterase activity of flavonoids and a significant correlation between the anticholinesterase activity of the studied Iranian EEPs and their total flavonoid content was observed, the interaction of 17 well-known propolis flavonoids with AChE and BuChE was explored using molecular docking. The results indicated that all the flavonoids interact with the active site gorge of both enzymes with high affinity. Summing up, the obtained results suggest that Iranian propolis possesses great potential for further studies.

Effects of synonymous mutations on kinetic properties and structure of firefly luciferase: Molecular dynamics simulation, molecular docking, RNA folding, and experimental study.

Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.

Modeling and optimization of radish root extract drying as peroxidase source using spouted bed dryer.

The main advantages of the dried enzymes are the lower cost of storage and longer time of preservation for industrial applications. In this study, the spouted bed dryer was utilized for drying the garden radish (Raphanus sativus L.) root extract as a cost-effective source of the peroxidase enzyme. The response surface methodology (RSM) was used to evaluate the individual and interactive effects of main parameters (the inlet air temperature (T) and the ratio of air flow rate to the minimum spouting air flow rate (Q)) on the residual enzyme activity (REA). The maximum REA of 38.7% was obtained at T = 50 °C and Q = 1.4. To investigate the drying effect on the catalytic activity, the optimum reaction conditions (pH and temperature), as well as kinetic parameters, were investigated for the fresh and dried enzyme extracts (FEE and DEE). The obtained results showed that the optimum pH of DEE was decreased by 12.3% compared to FEE, while the optimum temperature of DEE compared to FEE increased by a factor of 85.7%. Moreover, kinetic parameters, thermal-stability, and shelf life of the enzyme were considerably improved after drying by the spouted bed. Overall, the results confirmed that a spouted bed reactor can be used as a promising method for drying heat-sensitive materials such as peroxidase enzyme.

Immobilization of Lepidium draba peroxidase on a novel Zn-MOF nanostructure.

In the present study, ultrasound irradiation was utilized to synthesize a novel zinc metal-organic framework (MOF). Scanning electron microscopic images, exhibited homogenous morphology with a nano-sized distribution of the Zn-MOF structure as also confirmed by X-ray diffraction patterns. Following, physical immobilization of Lepidium draba peroxidase (LDP) were optimized on the Zn-MOF in phosphate buffer (50 mM, pH 6.5), ratio amount of MOF/enzyme; 7/1 after shaking for 15 min at 25 °C, with high protein loading of 109.9 mg/g and immobilization yield of 93.3%. Immobilized enzyme (IE) exhibited more than 330% enhanced specific activity and also exhibited more than 150% specific affinity to its substrate (3,3',5,5'-tetramethylbenzidine) with respect to the free enzyme (FE). Optimum temperature of the IE was obtained at 20 °C while its was 25 °C for the FE, and thermostability of the IE augmented at temperature of 30 °C and 40 °C by the factors of 104 and 108% respectively. pH stability under neutral and basic condition and storage stability of the IE improved with respect to the FE as well as its structural stability (Tm; 73 °C for IE vs. 63 °C for FE). Furthermore, immobilization is accompanied with alteration on the enzyme structure as revealed by the intrinsic and extrinsic fluorescence spectra.

Upregulation of pluripotent long noncoding RNA ES3 in HER2-positive breast cancer.

Breast cancer is the second most common cancer and estimates to be responsible for 20% of all cancer patients. Breast cancer has several subtypes including luminal A, luminal B, normal breast-like, basal-like, and human epidermal growth factor receptor 2 (HER2)-enriched. HER2-positive breast cancer cells have higher HER2 expression than other breast cancer subtypes. This subtype is the most aggressive breast cancer subtype and has more ability to metastasis than other breast cancer subtypes. HER2 is a growth-promoting protein that is overexpressed in approximately 20 to 30% of breast cancers and its overexpression is strongly related to poor prognosis. New studies suggested that HER2 expression is correlated with cancer stem cell (CSC) markers in breast cancer. ES3 transcript as a pluripotency long noncoding RNA (lncRNA) is linked to pluripotency transcriptional networks in human embryonic stem cells, but its function in breast cancer is not clarified. In the current research, we found ES3 upregulation in breast cancer and its diagnostic value in breast cancer diagnosis. Furthermore, our findings revealed that ES3 transcript has a high expression in high-grade and high-stage breast tumors. In addition, our data demonstrated that ES3 expression downregulated during neural differentiation. Therefore, its expression may be correlated to breast tumor differentiation status. Notably, a high expression level of ES3 in HER2-positive breast tumor tissues motivated us to investigate the effect of HER2 on ES3 expression by blocking HER2 activity with lapatinib. The results showed that ES3 expression suppressed when HER2 activity was blocked. In summary, for the first time, we found that lncRNA ES3 was significantly upregulated in HER2-positive breast tumors and may contribute to breast cancer proliferation as a downstream target of HER2.

Improvement of kinetic properties and thermostability of recombinant Lepidium draba peroxidase (LDP) upon exposed to osmolytes.

In this study, effects of different concentrations of glycine and D-sorbitol were analyzed on the activity and thermostability of recombinant Lepidium draba peroxidase (LDP). Based on the results, activity of the enzyme increased in the presence of various concentrations of these osmolytes. Maximum activity was detected for the enzyme in the presence of 300 mM glycine and 600 mM sorbitol. In presence of the aforementioned doses of osmolytes, enzyme affinity for substrate (3,3',5,5'-tetramethylbenzidine and H2O2) and Vmax increased. According to the results, enzyme stability improved against temperature and H2O2. Furthermore, structural changes of the enzyme upon exposure to the osmolytes were revealed by the use of far-UV circular dichroism and fluorescence methods. The results showed, whereas the secondary structure of the enzyme was not significantly changed upon exposed to the osmolytes, the fluorescence studies revealed microenvironment of the aromatic residues dramatically affected by them. Overall, it may be speculated, structural changes of the enzyme upon exposed to the osmolytes, lead to the improvement of its kinetic properties and stability that can be benefit for using of it in in vitro applications.

Optimization of in vitro refolding conditions of recombinant Lepidium draba peroxidase using design of experiments.

The main objective of this study was to optimize the in vitro refolding conditions of the recombinant Lepidium draba peroxidase (LDP). Initially, the effects of various factors were investigated on LDP refolding yield using one-factor-at-a-time (OFAT) method. Based on the OFAT results, optimum concentrations for LDP refolding were 2 M urea, 2 mM CaCl2, 0.42 mM l-glutathione oxidized (GSSG), 0.20 mg/ml protein, and 12 µM hemin as well as pH 7. Secondly, according to the OFAT results, design of experiments (DOE) was applied for investigation of the interactions between factors including protein (P), urea (U), CaCl2 (C), and GSSG (G). The results showed the possible interaction between PC, PG, PU, and GU. Lastly, response surface methodology (RSM) was used for final refolding conditions optimization. The final optimized refolding conditions for LDP were conducted as 2 M urea, 1 mM CaCl2, 0.70 mM GSSG, 0.07 mM DTT, 0.15 mg/ml protein which they obtained from RSM results and 12 µM hemin, and pH 7 according to the results of OFAT method. Overall, under optimal conditions, 23.4 mg active refolded LDP per liter of expression medium was obtained. So, the refolding yield was calculated to be approximately 48%.

Heterologous Expression, Purification and Characterization of a Peroxidase Isolated from Lepidium draba.

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.

In silico locating the immune-reactive segments of Lepidium draba peroxidase and designing a less immune-reactive enzyme derivative.

Peroxidases have broad applications in industry, environmental as well as pharmaceutical and diagnosis. Recently applicability of peroxidases in cancer therapy was mentioned. In the present study, a horseradish peroxidase homologue from Lepidium draba was subjected to in silico analyzes aiming at identifying and locating immune-reactive regions. A derivative sequence with decreased immunogenicity and increased stability also suggested. The tertiary structure of the enzyme was predicted. The functional and structural importance of residues was annotated as well as the conservatory status of each residue. The immune-dominant regions of protein were predicted with various software. N-terminal 4 residues, NFSHTGL (186-192), PRNGN (210-214), PLVRAYADGTQKFFN (261-275), and last 4 residues in C-terminal were predicted to be the consensus immunogenic segments of L. draba peroxidase. The modifications were applied to wild type sequence in order to mitigate its immune-reactiveness. The modifications were based on predicted energetic status of residues and naturally occurred amino acids in each position of the enzyme sequence, extracted from alignment file of 150 homologous peroxidases. The new enzyme derivative is predicted to be less immune-reactive and more stable. Thus the sequence is better suited to therapeutic applications.

Toxicity of essential oil of Satureja khuzistanica: in vitro cytotoxicity and anti-microbial activity.

In nature, essential oils play an important role in the protection of the plants by exerting anti-bacterial, -viral, -fungal, -oxidative, -genotoxic, and free radical scavenging properties, as well as in some cases acting as insecticides. Several Satureja species are used in traditional medicine due to recognized therapeutic properties, namely anti-microbial and cytotoxic activities. The purpose of the present work was to determine the biologic activity of the essential oil of S. khuzistanica Jamzad (Lamiaceae) against four human cancer cell lines, as well as its inhibitory effects against a wide array (i.e. n = 11) of pathogenic bacteria and fungi. The essential oil was isolated by hydro-distillation and analyzed by GC-FID and GC-MS. Carvacrol (92.87%) and limonene (1.2%) were found to be the main components of the isolated oil. Anti-microbial activity of the essential oil was assessed using a disc diffusion method; an MTT cytotoxicity assay was employed to test effects of the oil on each cancer cell line. The oil exhibited considerable anti-microbial activity against the majority of the tested bacteria and fungi. The test oil also significantly reduced cell viability of Vero, SW480, MCF7, and JET 3 cells in a dose-dependent manner, with the IC50 values calculated for each cell type being, respectively, 31.2, 62.5, 125, and 125 µg/ml. Based on the findings, it is concluded that the essential oil of S. khuzistanica and its major constituents have a potential for further use in anti-bacterial and anti-cancer applications, pending far more extensive testing of toxicities in normal (i.e. primary) cells.

Implication of Arg213 and Arg337 on the kinetic and structural stability of firefly luciferase.

Possible roles of two different Arginine (Arg; R) 213 and 337 on kinetic and structural stability of Photinus pyralis luciferase have been investigated using thermal and chemical denaturation studies. This enzyme is highly sensitive to protease digestion and temperature, which limits its fieldability, particularly for in vivo imaging. In order to generate more stable luciferases against trypsin digestion, site-directed mutagenesis was conducted to block two representative tryptic sites on the surface of N-terminal domain, via substitution of Arg213 and Arg337 by methionine (Met; M) and glutamine (Gln; Q), respectively [A. Riahi-Madvar, S. Hosseinkhani, Protein engineering, design and selection 22 (2009) 655-663]. The improvement of mutant enzymes stability against protease hydrolysis may be attributed to the more rigidity of the enzyme structure upon mutations, as can be deducted from elevated levels of m(U-N) values and decrease of activation energy. Furthermore, mutation at position 337 which is accompanied with more alteration on the basic kinetic properties relative to mutation at position 213, revealed the high values of the ΔG(H(2)O), half-time of inactivation at 30°C and T(m) for R337Q where Arg213 is maintained in structure. Based on the results, it can be concluded that whilst Arg213 affects structural stability, Arg337 is critical for kinetic stability.

Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis.

Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R(213), R(337) and also next residue to it (Q(338)) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging.