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Hyaluronidase (HAase) from bovine testes (BTH) has long been used in broad pharmaceutical areas, while it is associated with drawbacks in aspects of solubility, immunogenicity and pharmacokinetics. These issues can be addressed by gaining structural insights and designing rational modifications to the enzyme structure, as proposed in this study. A 3D structural model was built for HAase and underwent 40 ns of molecular dynamic simulation to examine its thermostability under normal, melting, and extreme conditions. The enzyme activity of BTH was measured against temperature and pH by kinetic assays. The interaction of bovine HAase with HA and chondroitin was defined by molecular docking. Furthermore, immunogenic properties of the enzyme were explored by immunoinformatics. Thermal effects on bovine HAase structural model and the HAase interactions with its substrates were described. We identified some B- and T-cell epitopes and showed that the protein could be recognized by human immune receptor molecules. Epitope masking by adding polyethylene glycol (PEG) to amine groups of residues presenting on the surface of the protein structure was adopted as a surface modification to enhance pharmacological properties of BTH. Assays showed that PEGylated BTH had higher thermostability and similar activity compared to the native enzyme.

Inflammation and the pro-inflammatory cytokines are associated with numerous chronic diseases. Studies suggest that flavonoids, plant polyphenolic compound derivatives from natural origin, have a wide range of putative biological activities. Similar to other flavonoids, chrysin (CH) by its anti-oxidative and anti-inflammatory effects is a potential prophylactic agent in immunopathological and physicochemical injuries. This is an overview on putative immunomodulatory activities of flavonoids and beneficial health effects of these substances particularly, CH in the immune system. CH possesses potent immune-protective effects and suppresses inflammation in innate immune system which results to avoid damages induced by neutrophils and macrophages and suppresses immuno-inflammatory responses. Furthermore, beneficial effects of chrysin on inhibition of serum levels nuclear transcription factor κB (NF-κB) p65 unit, tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-12, IL-17A, interferon gamma (IFN-γ) was reported. Moreover, CH has been known as the antagonist of NF-kB and the agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ) which in down regulation of the key pro-inflammatory enzymes such as myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), phospholipase A2, and prostanoids. Therefore, CH can improve immune system and neurodegenerative diseases. In recent years, there has been an increasing interest in compound derivatives from natural origin. Taken together, evidences show that flavonoids may have health-promoting and disease-preventing dietary compounds with important benefits in modern life styles.

A decrease in high-density lipoprotein (HDL) level and an increase in atherogenic low-density lipoprotein (LDL) concentration in plasma are involved in the progress of atherosclerosis and incidence of cardiovascular diseases. Cholesteryl ester transfer protein (CETP) passes cholesteryl ester and triglyceride molecules through a hydrophobic tunnel between lipoproteins and via that decreases the HDL/LDL ratio in plasma. Thus, inhibition of CETP activity by specific inhibitors could diminish atherosclerosis occurrence. Anacetrapib as a specific reversible inhibitor of CETP by stabilizing CETP–HDL complex significantly increases the HDL/LDL ratio in plasma. For better understanding the mechanism of CETP inhibition by Anacetrapib, after docking Anacetrapib against a CETP structure, molecular dynamics simulation of CETP and CETP–Anacetrapib in an explicit water cage was performed. Analyses of acquired trajectories show that Anacetrapib increases fluctuation of residues 421–442 of CETP through hydrophobic interactions and thus disrupts the integrity of an α-helix (helix B) structure in the C-terminal concave surface. Creation of a hinge by defeat of the helix B in the C-terminal of CETP, where CETP interacts with LDL, results in missing of an α-helix (residues 436–440) and conversion of banana-shaped CETP to a hemisphere conformation. According to the obtained results, bending of the CETP structure and conversion of the secondary structure in the C-terminal domain in the presence of Anacetrapib may destabilize CETP–VLDL complex and through that shift the dynamics of CETP to the formation of the CETP–HDL complex.

BACKGROUND Cellulose as a renewable biomaterial has forced attention on the use of cellulose-hydrolyzing enzymes for industrial bioconversion of lignocellulosic materials to glucose. This paper reports immobilization of cross-linked cellulase aggregates (CLEA) on the amine-functionalized Fe3O4@silica core-shell magnetic nanoparticles (MNPs). RESULTS The optimum pH of the cellulase cocktail upon immobilization (cellulase CLEA–MNP) shifted a little to the acidic side whereas the optimum temperature did not change significantly. The behavior of CMCase activity in the cellulase CLEA–MNP at pH and temperature values higher than the optimum was significantly different compared with free cellulase. Cellulase CLEA–MNP retained about 45% of its maximum activity at pH values higher than 4.8, while free cellulase lost its activity sharply. Immobilized cellulase in contrast to the free form retained about 65% of its maximum activity at 80°C. Cellulase CLEA–MNP had improved thermal stability at 65°C. Operational stability of the immobilized cellulase was also noticeable. After a sharp decrease during two cycles of CMC hydrolysis, cellulase CLEA–MNP retained 30% of its initial activity through six cycles of reuse. CONCLUSION Simple separation of MNPs from reaction medium and durability of CLEA–MNP composite during repeated use may overcome the major bottleneck against comprehensive applications of cellulase enzymes in industry. © 2014 Society of Chemical Industry

Iron overload in the amount of 10–20 mg/kg of body weight can induce toxicity. Iron causes its toxic effects in a form of oxidative stress and inhibition of key metabolic enzymes. It seems that montmorillonite clay can reduce absorption of elemental iron by iron fixation in the gastrointestinal tract. The potential effect of montmorillonite in decontaminating the gastrointestinal tract was investigated based on serum iron concentration. In the present experiment, ferrous sulfate (100 mg/kg) was administered to Wistar male rats followed by oral gavage of montmorillonite suspensions at three different doses (0.5, 1.0 and 1.5 g/kg). Blood for determination of serum iron concentrations was drawn at 1 h after montmorillonite administration. The results showed that montmorillonite clay with doses of 0.5 and 1.0 g/kg were the best for significantly decreasing the elevated serum iron concentration compared to 1.5 g/kg ( p

Trastuzumab (Herceptin(®) ) is a monoclonal antibody (mAb) for specific ablation of HER2-overexpressing malignant breast cancer cells. Intensification of antiproliferative activity of trastuzumab through construction of immunotoxins and nano-immunoconjugates is a promising approach for treatment of cancer. In this study, trastuzumab was directly conjugated to diphtheria toxin (DT). Also, conjugates of trastuzumab and multiwalled carbon nanotubes (MWCNT) were constructed by covalent immobilization of trastuzumab onto MWCNTs. Then, antiproliferative activity of the fusion constructs against HER2-overexpressing SK-BR-3 and also HER2-negative MCF-7 cancer cell lines were examined. Cells treated with trastuzumab-MWCNT conjugates were irradiated with near-infrared (NIR) light. Efficient absorption of NIR radiation and its conversion to heat by MWCNTs can be resulted to thermal ablation of cancerous cells. Our results strongly showed that both trastuzumab-MWCNT and trastuzumab-DT conjugates were significantly efficient in the specific killing of SK-BR-3 cells. Targeting of MWCNTs to cancerous cells using trastuzumab followed by exposure of cells to NIR radiation was more efficient in repression of cell proliferation than treatment for cancer cells with trastuzumab-DT. Our results also showed that conjugation linkers can significantly affect the cytotoxicity of MWCNT-immunoconjugates. In conclusion, our data demonstrated that trastuzumab-MWCNT is a promising nano-immunoconjugate for killing of HER2-overexpressing cancerous cells.

Malfunction or overexpression of ErbB receptors (epidermal growth factor receptors) is associated with occurrence and severity of several types of cancer. Initiation of signal cascades by ErbB2 (also known as human epidermal growth factor receptor 2/neu) in breast cancer has been blocked by monoclonal antibodies such as Trastuzumab (Herceptin), which interact with the extracellular domain of human epidermal growth factor receptor 2. Due to some disadvantages of monoclonal antibodies, a new approach in blocking human epidermal growth factor receptor 2 dimerization and activation is designing peptidomimetic ligands based on human epidermal growth factor receptor 2-Trastuzumab interaction model. Growth of human epidermal growth factor receptor 2(+) SK-BR3 cells could be specifically inhibited by peptidomimetic herceptin-based peptidomimetic 5. In this study, herceptin-based peptidomimetic 5 was used as a benchmark peptidomimetic compound to perform a ligand-based virtual screening followed by structure-based screening by applying the molecular docking approach. The study aimed to explore more potent peptidomimetic molecules against extracellular part of human epidermal growth factor receptor 2. Our results showed that the newly designed peptidomimetic herceptin-based peptidomimetic n33B in comparison with herceptin-based peptidomimetic 5 binds more tightly to the extracellular domain of human epidermal growth factor receptor 2. Mechanistic aspects of herceptin-based peptidomimetic n33B interaction with human epidermal growth factor receptor 2 were more investigated through 20-ns molecular dynamic simulations. Additionally, a quantitative structure-activity relationships study was performed to develop a model for identification of structural determinants in herceptin-based peptidomimetic 5-based peptidomimetics.

Tuberculosis that is caused by Mycobacterium tuberculosis is one of the widespread infectious diseases with millions of death worldwide. Prevalence of Multi-drug-resistant (MDR) tuberculosis and emergence of extensively drug-resistant (XDR) species of M. tuberculosis have necessitated the discovery of new and potent antibiotics. Designing of specific inhibitors for 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS) has attracted a great interest because of its crucial role in de novo folate synthesis in prokaryotic but not in mammalian cells. In the current study, the ligand of DHPS, namely 6-hydroxymethylpterin monophosphate (PtP) was used as a template for structural-based inhibitor design. Virtual screening was performed between compounds with structural similarity to PtP. Between docked compounds against DHPS, 2-amino-6-(1,2,3-trihydroxybutyl)-1H-pteridin-4-one (PtP_1) showed the most negative free energy of binding. Binding of PtP and PtP_1 to DHPS was further investigated through molecular dynamic simulation. DHPS as alone or in complex with ligands was minimized, solvated in an explicit water cage, neutralized by addition of required ions and simulated for 50 ns. According to trajectories, PtP_1 shows high affinity for the active site of DHPS by H-bonds, hydrophobic forces, and cation-? interactions. Our results show that there could be a great chance for designing new inhibitors against DHPS from M. tuberculosis .

Lactate dehydrogenase C (LDHC) as the most abundant isoform of lactate dehydrogenase in testis and spermatozoa, has a critical role in energy metabolism of germ cells. Also, LDHC gene is expressed aberrantly with a high prevalence in various kinds of malignancies. So, LDHC is a promising target protein for development of contraceptive and anti-cancer drugs. In the current study, inhibitory effects of nicotine and cotinine on human LDHC were investigated. Partially purified LDHC was extracted from healthy human spermatozoa and treated with various concentrations of nicotine and cotinine at a range of 0.13-0.66 μM. The results showed that at a concentration of 0.6 μM, cotinine and nicotine could inhibit LDHC activity by 47% and 45%, respectively. Mechanisms of these inhibitory effects were further investigated by docking and molecular dynamic (MD) simulation studies. Nicotine and cotinine were docked against available crystal structure of mice LDHC (PDB ID: 2LDX). LDHC alone and in complex with docked ligands was minimized, solvated in explicit water cage, neutralized by addition of required ions, and finally simulated for 50 ns of MD simulation. Analysis of simulated structures revealed that nicotine and cotinine could affect LDHC structure near His192 which play a key role in the interaction of LDHC with pyruvate/lactate. Thus, it seems that nicotine and cotinine could induce their effects via an inhibitory competition mechanism. Enzymatic assays with various concentration of pyruvate in the presence of mean plasma concentration of nicotine and cotinine in active smokers confirmed MD simulation results.

Microperoxidase-11 (also known as MP-11) was chemically modified by boron trifluoride–methanol reagent to obtain methylated MP-11 (mMP-11). Porphyrin transformation capability of mMP-11 in a ternary solvent system was investigated by spectroscopic and kinetic techniques and compared with those of native MP-11. Methyl-modified MP-11 showed higher porphyrin transformation efficiency for synthetic nickel- and vanadium-porphyrins than native MP-11. Upon the chemical modification, the initial rates of porphyrin transformation in a solvent mixture of methylene chloride, methanol and water (up to 5%, v/v) were increased by 60.0-, 4.1-, 7.0- and 1.13-fold for VTPP (V-tetraphenyl porphyrin), VOEP (V-octaethyl porphyrin), NiTPP (Ni-tetraphenyl porphyrin) and NiOEP (Ni-octaethyl porphyrin), respectively. Modified MP-11 also exhibited a good degradation potential in the presence of kerosene (a crude oil fraction). In an organic solvent mixture containing kerosene (35%, v/v), mMp-11 in compared to unmodified MP-11 showed 46.0-, 2.1-, 14.1- and 3.1-times higher activity for VTPP, VOEP, NiTPP and NiOEP, respectively. Experimental data showed that concentration and type of organic hydroperoxides, temperature, and pH of the added water play significant roles on the porphyrin degradation.

An electrochemical immunosensing method was developed based on a magnetic nanocomposite. The multiwalled carbon nanotubes (MWCNTs) were treated with nitric acid to produce carboxyl groups at the open ends. Then, Fe₃O₄ nanoparticles were deposited on COOH-MWCNTs by chemical coprecipitation of Fe²⁺ and Fe³⁺ salts in an alkaline solution. Goat anti-human IgG (anti-hIgG) was covalently attached to magnetic nanocomposite through amide bond formation between the carboxylic groups of MWCNTs and the amine groups of anti-hIgG. The prepared bio-nanocomposite was used for electrochemical sensing of human tetanus IgG (hIgG) as a model antigen. The anti-hIgG magnetic nanocomposite was fixed on the surface of a gold plate electrode using a permanent magnet. The hIgG was detected using horseradish peroxidase (HRP)-conjugated anti-hIgG in a sandwich model. Electrochemical detection of hIgG was carried out in the presence of H₂O₂ and KI as substrates of HRP. Using this method, hIgG was detected in a concentration range from 30 to 1000 ng ml⁻¹ with a correlation coefficient of 0.998 and a detection limit of 25 ng ml⁻¹ (signal/noise=3). The designed immunosensor was stable for 1 month.

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-gamma secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 microg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-gamma and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 microg glycan/ml.

Aims: The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined. Methods and Results: When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2′-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o-hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites. Conclusions: Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho-cleavage of the 9,10-diol leads to formation of 2,2′-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o-phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho-cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene. Significance and Impact of the Study: For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.

The thermophilic bacterium Nocardia otitidiscaviarum strain TSH1, originally isolated in our laboratory from a petroindustrial wastewater contaminated soil in Iran, grows at 50 degrees C on a broad range of hydrocarbons. Transformation of naphthalene by strain TSH1 which is able to use this two ring-polycyclic aromatic hydrocarbon (PAH) as a sole source of carbon and energy was investigated. The metabolic pathway was elucidated by identifying metabolites, biotransformation studies and monitoring enzyme activities in cell-free extracts. The identification of metabolites suggests that strain TSH1 initiates its attack on naphthalene by dioxygenation at its C-1 and C-2 positions to give 1,2-dihydro-1,2-dihydroxynaphthalene. The intermediate 2-hydroxycinnamic acid, characteristic of the meta-cleavage of the resulting diol was identified in the acidic extract. Apart from typical metabolites of naphthalene degradation known from mesophiles, benzoic acid was identified as an intermediate for the naphthalene pathway of this Nocardia strain. Neither phthalic acid nor salicylic acid metabolites were detected in culture extracts. Enzymatic experiments with cell extract showed the catechol 1,2-dioxygenase activity while transformation of phthalic acid and protocatechuic acid was not observed. The results of enzyme activity assays and identification of benzoic acid in culture extract provide strong indications that further degradation goes through benzoate and beta-ketoadipate pathway. Our results indicate that naphthalene degradation by thermophilic N. otitidiscaviarum strain TSH1 differs from the known pathways found for the thermophilic Bacillus thermoleovorans Hamburg 2 and mesophilic bacteria.

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.