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Tyrosinase also known as polyphenol oxidase (PPO) is a copper-containing mono-oxygenase, which is responsible for melanization in animals and the enzymatic browning of fruits. It displays two distinct enzymatic activities: hydroxylation of monophenols to o-diphenols (monophenolase activity) and oxidation of the latter to o-quinones (diphenolase activity). The inhibition of tyrosinase is very important and encourages us to takes special attempts to search for new inhibitors. For the first time in the present study, the effects of 2-nitroaniline (a), 3-nitroaniline (b) and 4-nitroaniline (c) as well as their newly synthesized vanillin derivatives (2-nitrobenzenaminium 4-formil-2-metoxyphenolate (d), 3-nitrobenzenaminium 4-formil-2- metoxyphenolate (e) and 4-nitrobenzen aminium 4-formil-2-metoxyphenolate (f)) were studied on the oxidation of dopamine hydrochloride by mushroom tyrosinase. Among them, 4-nitroaniline (c) exhibited the strongest inhibitory effect, while acted as an activator. For these compounds, the IC50 follows the order of b> f= a> e> c. Compounds a, b and f were competitive while c and e were un-competitive inhibitors. The results indicated that the relative positioning of amino and nitro groups is important in the inhibition of the enzyme.

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 Maltogenic Amylases (MAase) are a subfamily of Į-amylase family that can hydrolyze multiple substrates including starch, pullulan and cyclodextrins however, they prefer cyclodextrins to others, and unlike other Į-amylases, they are intracellular. This enzyme has the potential for use in many industrial processes such as food, fermentation and pharmacy. The effect of different concentrations of Ca2+ and K+ ions on irreversible thermoinactivation of the enzyme at 65 ÛC showed that Ca2+ and K+ decreased and increased its thermal stability. The CD spectra of the enzyme in the presence and absence of metal ions were measured to detect changes in the secondary structure contents. The spectra showed a decrease in the Į-helix content in the presence of 1 and 10 mM of Ca2+, but in the presence of 5 mM, a drastic increase in Į-helix content of the enzyme was witnessed. In the presence of 1 and 5 mM of Na+ the Į-helix content decreased, while it was increased in the presence of 10 mM. The results from intrinsic fluorescence of the protein (excitation at 280 nm) indicated that Ca2+ ion at 1 and 5 mM caused an increase in tertiary structure of the enzyme; however, at 10 mM, a decrease was observed in its tertiary structure. K+ ion at all concentrations increased the tertiary structure of the enzyme. These spectroscopic results are in a good agreement with the thermostability data. It was shown that destabilizing effect of calcium was enthalpic (decrease in ǻH#) whereas the stabilizing effect of potassium was entropic (decrease in ǻS#).

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Mnemiopsin, a Ca2+-regulated photoprotein from ctenophore Mnemiopsis leidyi, as coelentrate photoproteins emits flash blue light upon reacting with coelenterazine. In contrast to coelenterate photoproteins, there is a little information about the structure of chromophore binding site and bioluminescence mechanism in ctenophore photoproteins. In this study, three important amino acid residues in coelenterazine binding cavity of mnemiopsin were substituted by corresponding residues in the well-known coelentrate photoproteins. W59K, N105W and L127W mutants were constructed and characterized for investigation of hydrogen bond network around the important rings of coelenterazine. All three mutants are completely inactivated. In addition, the results of structural studies including CD, intrinsic and extrinsic fluorescence together with theoretical studies showed that these mutants, especially for N105W and L127W, have found different structural features. These results suggest the presence of the residues in binding cavity and/or a mechanistic role for these residues. It seems that arrangement of amino acid residues in the binding cavity of coelenterate and ctenophore photoproteins are different, so that the replacement of these residues with their corresponding residues in other group (such as mutations in this study) perturbs the structural integrity needed for bioluminescence activity.

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Regarding the importance of inhibiting VEGF and unique features of VHHs as a new generation of antibody-based therapeutics, the present study aimed to generate VHHs against the receptor binding domain of VEGF, thereby blocking of VEGF binding to its receptor. After preparing the gene repertoire of VHH fragments from an immunized camel, a VHH phage display library was constructed. We adopted a stringent successive biopanning to isolate the phages displaying VHH with high affinity to VEGF-RBD.A significant enrichment of phages that specifically bound to the target protein was obtained after six rounds of panning. Of the specific clones with high binding affinity screened by monoclonal phage ELISA, 52% shared the same VHH sequence, showing its high enrichment. Using molecular simulation of antigen-antibody interaction based on the crystallographic information of VEGF/VEGFR2, molecular dynamics simulations and MM/PBSA free energy calculations, we provide a reliable picture of the binding site of antibody on antigen. The key residues in the VEvhh1-VEGF interface were dissected and the energetics was analyzed by MM/PBSA. The results of studies revealed that VEvhh1 binds to the receptor binding site of VEGF with high binding energy and showed the highest affinity to the residues of VEGF which are responsible for VEGF binding to VEGFR2. Also the antibody potently covers these key functional residues of VEGF, thereby inhibiting VEGF binding to its receptor and probably abrogating its biological activity. This study may represent VEvhh1 as an anti-VEGF and anti-angiogenic candidate.

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The toxicological and biochemical properties of four organophosphate (OP) insecticides, chlorpyrifos, diazinon, phosalone and dichlorvos, were examined in terms of the diamond back moth, Plutella xylostella (L.) susceptible (Gu-S) and resistant (Kar-R) to OPs. The Kar-R population had significantly high resistance to chlorpyriphos (69.3 fold), medium resistance to diazinon (14.49-fold) and phosalone (10.3-fold), and had less resistance to dichlorvos (5.17-fold) compared to Gu-S population. DEM and TPP reduced Chlopyrifos resistance of Kar-R population as an inhibitor of glutathione S-transferase (GST) and esterases (EST), respectively. Biochemical studies clarified that GST and EST kinetic parameters in the Kar-R population were significantly higher than parameters of Gu-S population. Moreover, this study examined the Kinetics of hydrolysis of acetylthiocholine iodide, butyrylthiocholine iodide as artificial substrates by AChE of resistant and susceptible population. IC₅₀ of monocrotophos, neostigmine bromide and eserine were also determined on AChE of resistant and susceptible populations. Kinetic analysis and inhibition tests indicated that an alteration in AChE of Kar-R population has an effect on both kinetic and inhibition results. The results distinctly showed that multiple mechanisms such as GST, esterases and altered AChE created chlorpyrifos resistance in the Kar-R and insensitivity of AChE is a significant factor for resistance to conventional OP compounds.
 

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Abnormal angiogenesis is associated with various diseases such as solid tumors and metastasis, retinopathies, and rheumatoid arthritis. VEGF-A is the most important mediator of angiogenesis among all growth factors. The bioactivity of VEGF is mediated by two tyrosine kinase receptors VEGFR-1 and VEGFR-2 present on endothelial cells. VEGF signaling through VEGFR-2 is the major angiogenic pathway that leads to stimulation of endothelial cell ingrowths into the tumor. It comprised of an extracellular portion, a cytoplasmic portion, and a short transmembrane domain. The extracellular portion consists of seven Ig-like domains (D1–D7), of which the 1st to 3rd domains function as ligand-binding sites. In the present work, a soluble recombinant extracellular domain 1-3 of VEGFR-2 was expressed in Pichia pastoris to inhibit the VEGF-induced angiogenesis. The 975 bp DNA fragment containing extracellular domain 1-3 kdr, was designed according to the nucleotide sequence at GenBank and protein sequence at Swiss-Prot. The recombinant secretory expression vector (pPinkαHC/KDR1-3) was constructed and transferred into yeast by electroporation. The high expression transformants were identified through complementation of adenine auxotrophy and cultured. KDR1-3 was expressed under the induction of %1 methanol and confirmed by using SDS-PAGE and western blot techniques. After being purified by affinity chromatography using Ni-NTA resins, binding of expressed product to hVEGF165 was proved by two direct ELISA and ELISA receptor binding assays. The data showed that human VEGFR-2 extracellular domain 1-3 with eukaryotic protein structure, that there is no reported paper about, was successfully expressed. 

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Aim: In this study, the antioxidant properties of hydrolyzed protein from longtail tuna dark muscle with commercial enzymes (alcalase, alkaline protease, and evatase) were investigated.
Materials & Methods: Protein hydrolysates from tuna dark muscle were prepared by different enzymes Degree of hydrolysis (DH) was performed by TCA technique. The five aliquots at 60, 180, 240, 300, and 360 min were gathered during hydrolysis. The antioxidant activity of aliquots was monitored by in vitro assays (DPPH inhibition ability and Ferric (Fe3+) reducing power).
Findings: The antioxidant activities of protein hydrolysate from tuna dark muscle (TDM) increase with increasing time and DH. Alcalase hydrolyzed protein (AHP) generally showed higher antioxidative activity than evatase hydrolyzed protein (EHP) and alkaline protease hydrolyzed protein (APHP). Among the samples (concentration 3 mg.ml^-1), AHP at 360 min significantly exhibited the highest ability to scavenge DPPH radical (72.6 %). Furthermore, AHP and APHP significantly showed a minimum IC50 value of 1.1 mg.ml^-1 at 240 and 360 min hydrolysis. APHP significantly exhibited the highest ferric reducing power of 0.83 at 300 min and 0.76 at 240 min. AHP and APHP significantly showed the highest ferric reducing power of 0.74 at 360 min (p < 0.05).
Conclusion: This study confirmed that protein hydrolysate from TDM could be a good source of antioxidant peptides. In addition, the antioxidant activity of hydrolyzed protein relay on protease type and hydrolysis condition.
 

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Abstract. FZD7 receptor is considered as an emerging target for the treatment of Wnt-βcatenin related cancers. This transmembrane receptor is overexpressed in many cancer types like breast cancer and ovarian carcinoma, and so selective targeting of this receptor has a great therapeutic capacity. On the other hand, one of the mechanisms proposed for the anticancer effect of Atrial natriuretic peptide (ANP) that known as a heart hormone at first, is Wnt-βcatenin inhibition through an FZD dependent manner but, the molecular mechanism of this inhibition is not clear. Here, using computational methods including molecular docking and molecular dynamics simulation, also designing a cellular system that enabled us to trace Wnt-βcatenin kinetics directly, we investigated the mechanism of the peptide inhibitory potential against the pathway. Our computational results show that ANP can directly interact with FZD7 and also, its binding site on FZD7 overlap to the binding region of the Wnt carboxyl-terminal domain (Wnt-CTD). The finding of the silencing experiments demonstrates the dependency of Wnt-βcatenin signaling of the cellular system to FZD7. The decrease of βcatenin in cells treated to ANP and Wnt is also significant to compare to the control experiments. Finally, our results show that ANP is a potential scaffold to design selective peptide against FZD7.

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Transforming growth factor beta (TGF-β), is a small homodimeric signaling protein. The TGF-β isoforms (TGFβ1, β2 and β3) are involved in many cellular processes including growth inhibition, extracellular matrix remodeling, tissue development, cell migration, invasion and immune regulation. For research aims, TGFβs are overexpressed using recombinant eukaryotic cell or bacterial expression systems. For achieving an efficient purification of TGF-β by immobilized metal ion affinity chromatography (IMAC), a histidine tag was placed either at the C-terminal (C-TGFβ) or N-terminal (N-TGFβ) region of the sequence and the effect of His-tag on TGF-β structure has been studied by computational tools. Proteins 3D structures were modeled using MODELLER software and molecular dynamics simulation of native TGF-β and modelled proteins, N-TGFβ and C-TGFβ were studied in water by GROMACS package. Protein dynamics modeling indicated that the His-tag attached at the C-terminus but not at the N-terminus of the TGF-β can affect the fluctuations of amino acids and protein structure. It is concluded that the C-terminal tagging may cause distortion and misfolding in the structure.

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Investigation of factors affecting endothelial cell proliferation is an essential part of angiogenesis studies. Given the importance of inhibiting angiogenesis in the treatment of cancers, and due to the side effects and high cost of anti-angiogenic drugs such as Avastin, the use of physical agents to aid in treatment and reduce the need for high doses of the drug is noteworthy. Magnetic fields are of interest due to their long-distance and non-invasive effects, and many studies have been conducted on their effects on biological phenomena, including angiogenesis, with inconsistent results. In the present study, the effect of a 2 mT alternating magnetic field with a frequency of 200 Hz and Austin on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated. Cells were treated for 48 hours under a mixture of 50 μg/ml solution of vascular endothelial growth factor (VEGEF) and Avastin at concentrations (zero (drug control), 50, 100, 200 and 400 μg/ml) as well as field treatment groups for They were exposed to magnetic fields for 3, 6, 12, 24 and 48 hours. Then, cell proliferation was assessed using Alamar Blue colorimetric test. Data were analyzed by three-way analysis of variance. According to the findings, the exposure times of 12, 24 and 48 hours showed a significant reduction in cell proliferation compared to the control group, but this difference was not significant in the 3 and 6 hour treatments. Also, the degree of interaction of these factors with each other on HUVEC proliferation was investigated.

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