Publications by authors named "Bahareh Dabirmanesh"

32 Publications

CD38 and MGluR1 as possible signaling molecules involved in epileptogenesis: A potential role for NAD homeostasis.

Brain Res 2021 Aug 28;1765:147509. Epub 2021 Apr 28.

Department of Medical Physiology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis.
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http://dx.doi.org/10.1016/j.brainres.2021.147509DOI Listing
August 2021

Proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy: potential targets in calcium regulatory network.

Sci Rep 2021 Apr 15;11(1):8252. Epub 2021 Apr 15.

Department of Medical Physiology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.

Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.
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http://dx.doi.org/10.1038/s41598-021-87555-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050094PMC
April 2021

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Biochemistry (Mosc) 2021 Feb;86(2):190-196

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115, Iran.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The K and k values of 6.5 µM and 4.57 s, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.
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http://dx.doi.org/10.1134/S0006297921020073DOI Listing
February 2021

Cancer-associated-platelet-inspired nanomedicines for cancer therapy.

Wiley Interdiscip Rev Nanomed Nanobiotechnol 2021 Feb 3:e1702. Epub 2021 Feb 3.

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China.

Platelets, with hemostasis and thrombosis activities, are one of the key components in the blood circulation. As a guard, they rapidly respond to any abnormal blood vessel injury signal and release their granules' contents, which induce their adhesion and aggregation on wound site for hemostasis. Recently, increasing evidence has indicated that platelets are critically involved in the growth and metastasis of cancer cells by releasing a variety of cytokines and chemokines to stimulate cancer cell proliferation and various angiogenic regulators to accelerate tumor angiogenesis. Platelets also secrete active transforming growth factor beta (TGF-β) to promote the epithelial-mesenchymal transition of cancer cells and their extravasation from primary site, and form microthrombus on the surface of cancer cells to protect them from immune attack and high-speed shear force in the circulation. Therefore, blocking platelet-cancer cell interaction may be an attractive strategy to treat primary tumor and/or prevent cancer metastasis. However, systemic inhibition or depletion of platelets brings risk of severe bleeding complication. Cancer-associated-platelets-targeted nanomedicines and biomimetic nanomedicines coated with platelet membrane can be used for targeted anticancer drug delivery, due to their natural targeting ability to tumor cells and platelets. In the current review, we first summarized the platelet mechanisms of action in physiological condition and their multiple roles in cancer progression and conventional antiplatelet therapeutics. We then highlighted the recent progress on the design and fabrication of cancer-associated-platelet-targeted nanomedicines and platelet membrane coating nanomedicines for cancer therapy. Finally, we discussed opportunities and challenges and offered our thoughts for the future development. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures.
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http://dx.doi.org/10.1002/wnan.1702DOI Listing
February 2021

G-quadruplex binding protein Rif1, a key regulator of replication timing.

J Biochem 2021 Feb;169(1):1-14

Genome Dynamics Project, Department of Basic Medical Sciences, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, Japan.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.
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http://dx.doi.org/10.1093/jb/mvaa128DOI Listing
February 2021

Catalytic parameters and thermal stability of chondroitinase ABCI on red porous silicon nanoparticles.

J Biotechnol 2020 Dec 23;324:83-90. Epub 2020 Sep 23.

Tarbiat Modares University, Faculty of Biological Science, Tehran, Iran.

The bacterial enzyme chondroitinase ABC, which digests extracellular chondroitin sulfate proteoglycans, has been shown to enhance axonal regeneration. However, the utilization of this enzyme as therapeutics is notably restricted due to its thermal instability. Therefore, red luminescent porous silicon that hold promise for potential applications in biological/medical imaging was used as a carrying matrix for chondroitinase with the aim of enhancing its stability. Porous Si nanoparticles were prepared by electrochemical etching of silicon wafers in ethanolic HF solution. The size of nanoparticles (210 nm) and the mean pore diameter (8 -20 nm) were determined using dynamic light scattering and scanning electron microscopy. Purified chondroitinase was then incorporated into the silicon pores. Results revealed similar K and lower V value for the immobilized enzyme when compared with the free enzyme. The immobilized chondroitinase exhibited about a 4 fold increase in stability at 37 °C after 50 min. It is likely possible that, the enzyme was protected inside the pores resulted in higher stability. Moreover, porous silicon was seen to be capable of holding the chondroitinase for repeated cyclic tests for three times. The cell viability assay exhibited no significant cytotoxicity for Psi-chondroitinase up to 24 h.
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http://dx.doi.org/10.1016/j.jbiotec.2020.09.020DOI Listing
December 2020

Anti-amyloidogenic effect of artemin on α-synuclein.

Biol Chem 2020 09;401(10):1143-1151

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 μg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.
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http://dx.doi.org/10.1515/hsz-2019-0446DOI Listing
September 2020

A multifunctional fusion peptide for tethering to hydroxyapatite and selective capture of bone morphogenetic protein from extracellular milieu.

J Biomed Mater Res A 2020 05 18;108(7):1459-1466. Epub 2020 Mar 18.

VISTA Institute for Therapeutic Innovations, Woodland Hills, California, USA.

Purpose: The present study sought to design a multi-functional fusion peptide with hydroxyapatite (HA) binding domain (HABD) and heparin binding domain (HBD).

Methods: The 74 amino acid fusion peptide contained N-terminus of the fibrinogen β chain (β 15-66), double G4S-linker and 12 residues with HA affinity. This construct was designed, synthesized and cloned into pET21a(+) vector and expressed in E. coli.

Results: HABD facilitated purification of the fusion peptide by HA affinity chromatography. Kinetic peptide binding and release on HA scaffold showed sustained release of peptide for up to 16 days. Competitive ELISA and intrinsic fluorescence assays were applied to determine HBD affinity to bone morphogenetic protein-2 (BMP-2). The disassociation rate constant (K ) for HBD and rhBMP-2 was approximately 9.2-12 nM.

Conclusion: The fusion peptide developed in the present study, allowed for streamlined purification on HA affinity chromatography, as well as sustained release from HA scaffold, attributed to its HABD. HBD mediated binding to BMP-2, which may be potentially useful for bone repair. Additional studies, including in vivo investigation will be required to assess the efficacy of the fusion peptide in bone tissue engineering.
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http://dx.doi.org/10.1002/jbm.a.36915DOI Listing
May 2020

Enhancing myelin repair in experimental model of multiple sclerosis using immobilized chondroitinase ABC I on porous silicon nanoparticles.

Int J Biol Macromol 2020 Mar 31;146:162-170. Epub 2019 Dec 31.

Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address:

Removal of chondroitin sulfate proteoglycans (CSPGs) with chondroitinase ABC I (ChABC) facilitates axonal plasticity, axonal regeneration and remyelination, following injury to the central nervous system (CNS). However, the ChABC rapidly undergoes thermal inactivity and needs to be injected repeatedly. Here this limitation was overcame by immobilizing the ChABC on porous silicon (PSi) nanoparticles ([email protected]). The efficacy of immobilized ChABC on CSPGs level and the demyelination insult was assessed in mice corpora callosa demyelinated by 6 weeks cuprizone (CPZ) feeding. [email protected] was able to reduce the amount of CSPGs two weeks after animals treatment. CSPGs digestion by [email protected] reduced the extent of demyelinated area as well as the astrogliosis. Furthermore, [email protected] treatment increased the number of newly generated oligodendrocyte lineage cells which imply for enhanced myelin repair. Our results showed that effective CSPGs digestion by [email protected] enhanced remyelination in CPZ model. Accordingly, [email protected] may have a great potential to be used for treatment of diseases like multiple sclerosis and spinal cord injury by promoting the regeneration of damaged nerves.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.12.258DOI Listing
March 2020

Improvement of Selenomonas ruminantium β-xylosidase thermal stability by replacing buried free cysteines via site directed mutagenesis.

Int J Biol Macromol 2019 Sep 18;136:352-358. Epub 2019 Jun 18.

Department of Biochemistry, Neyshabur Branch, Islamic Azad University, Neyshabur, Iran. Electronic address:

β-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium β-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.06.100DOI Listing
September 2019

The interaction of several herbal extracts with α-synuclein: Fibril formation and surface plasmon resonance analysis.

PLoS One 2019 11;14(6):e0217801. Epub 2019 Jun 11.

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University,Tehran, Iran.

Proteins from their native conformation convert into highly ordered fibrillar aggregation under particular conditions; that are described as amyloid fibrils. α-Synuclein (α-Syn) is a small natively unfolded protein that its fibrillation is the causative factor of Parkinson's disease. One important approach in the development of therapeutic agents is the use of small molecules (such as flavonoids) that could specifically and efficiently inhibit the aggregation process. In this study the effect of few herbal extract (Berberis, Quercus robur, Zizyphus vulgaris, Salix aegyptica) containing flavonoids were investigated on fibril formation of α-syn by using conventional methods such as ThT fluorescence, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The interaction of extracts were also analysed by surface plasmon resonance (SPR). Among extracts, Salix aegyptica revealed the highest inhibitory effect on fibril formation. As expected, Salix aegyptica extract also exhibited the highest affinity toward α-syn. Cell viability using MTT assay revealed that fibrils alone were more toxic than those containing the extract. Overall, we demonstrated that the affinity of compounds used in this study corresponds to their ability to arrest fibrillation and reduce cellular toxicity of α-syn fibrils.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559707PMC
February 2020

Insight into the aggregation of lipase from Pseudomonas sp. using mutagenesis: protection of aggregation prone region by adoption of α-helix structure.

Protein Eng Des Sel 2018 11;31(11):419-426

Department of Biomedical, Experimental and Clinical Sciences, Section of Biochemistry, University of Florence, Viale Morgagni 50, Florence, Italy.

Previously, a lipase purified from a Pseudomonas source showed to form amyloid fibril structure very rapidly in the absence of a detectable lag phase. In this process the urea-unfolded enzyme encounters a medium close to physiological, but is unable to fold and, therefore, the main driving force of aggregation lies in the sequence of the protein and in its aggregation-promoting regions (APRs). Two regions with the highest propensity to aggregate were identified. These were Regions 51-57 and 160-172 as they were found with all four prediction algorithms. Two mutants of lipase, F171E and I52E, were selected and their propensity to aggregate was evaluated using thioflavin T (ThT), Congo red binding, circular dichroism, transmission electron microscopy (TEM) and dynamic light scattering. While I52E lipase formed aggregates that were capable of amyloid dye binding, showed a typical β-sheet structure and amorphous/fibrillar morphology, the aggregates formed by the F171E mutant indicated diminished ThT binding, lower light scattering, a smaller content of β-sheet structure and a lower presence of aggregates by TEM imaging. These data indicate that the region of the Sequence 160-172 is an APR region of this protein and lead to the suggestion of strategies aimed at promoting the solubility of this protein.
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http://dx.doi.org/10.1093/protein/gzz003DOI Listing
November 2018

Evaluation of angiotensin converting enzyme inhibitors by SPR biosensor and theoretical studies.

Enzyme Microb Technol 2019 Jan 23;120:117-123. Epub 2018 Oct 23.

Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, 14176-53955, Iran; Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, 14176-53955, Iran. Electronic address:

Surface plasmon resonance (SPR) biosensor has been utilized for monitoring analyte-ligand interactions in modern drug discovery processes. SPR biosensors measure the change in refractive indexes over the course of analyte molecules' binding to a specific immobilized ligand on sensor chip. This effort highlights a comprehensive SPR study besides enzymatic assay for discovery of new Angiotensin Converting Enzyme (ACE) inhibitors via screening of medicinal plants. At first, five medicinal plants were selected as potential sources for developing new ACE inhibitors through hydrolyzing hippuryl-L-histidyl-L-leucine (HHL) assay. The interaction of selected extracts with immobilized ACE on the sensor chip (500D) confirmed that the Onopordum acanthium L. had the greatest ACE inhibition activity among the set of compounds and its active compound (onopordia) was isolated. SPR biosensor used to evaluate binding affinity of onopordia and ACE. Equilibrium constant (K), and changes in Gibb's free energy of the binding (ΔG) values for the interaction of onopordia with ACE were found to be 10.24 μM and -28.48 kJ/mol, respectively. Computational analysis supported the binding of onopordia to the ACE active site. Kinetic and thermodynamic parameters of binding revealed that onopordia is an acceptable ACE inhibitor and could treat hypertension. SPR biosensor can be used to improve the drug discovery process for many important classes of drug targets due to its great sensitivity.
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http://dx.doi.org/10.1016/j.enzmictec.2018.10.010DOI Listing
January 2019

Study Break: Cell Timer/Cell Clock

Iran Biomed J 2018 11 16;22(6):360-1. Epub 2018 Sep 16.

Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamkitazawa, Setagaya-ku, Tokyo 156-8506, Japan.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305818PMC
November 2018

Decolorization of dyes by a novel sodium azide-resistant spore laccase from a halotolerant bacterium, Bacillus safensis sp. strain S31.

Water Sci Technol 2018 Jul;77(11-12):2867-2875

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v) methanol, ethanol, and acetone. In contrast to many other laccases, NaN, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.
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http://dx.doi.org/10.2166/wst.2018.281DOI Listing
July 2018

Improve Salinivibrio zinc-metalloprotease function in less polar organic solvents by increasing surface hydrophobicity.

Int J Biol Macromol 2018 Dec 18;120(Pt A):440-448. Epub 2018 Jul 18.

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

Organic solvents tend to strip water from protein and thereby disrupt non-covalent forces and decrease enzyme activity and stability. In the present study, we have replaced the surface charge residues in Salinivibrio zinc-metalloprotease (SVP) with hydrophobic ones (E12V, D22I, D24A and D310I) in order to study the effects of surface hydrophobicity with hydrophobic strength of organic solvents. Compared to SVP, D24A exhibited an increase in k and catalytic efficiency and a reduction in thermal inactivation rate in aqueous solvent. Structural studies indicated that the replacement of surface charge residues with hydrophobic residues would not induce conformational changes. C value (the value of solvent concentration where 50% of enzyme activity remains), k (irreversible thermoinactivation rate), and kinetic parameters of E12V, D22I, and D24A were higher in isopropanol and n-propanol. D24A is found to be the most efficient mutant for its remarkable decrease in k value in the presence of isopropanol and n-propanol and a reduction in k value in the presence of dimethylformamide (DMF) and methanol. C value in this variant was increased about 1.2% in DMF, 2% in methanol and isopropanol and 2.5% in n-propanol. Results revealed that, there was a correlation between surface hydrophobicity of SVP and hydrophobic strength of organic solvents.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.07.093DOI Listing
December 2018

Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Int J Biol Macromol 2018 Jun 6;112:775-779. Epub 2018 Feb 6.

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address:

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.02.019DOI Listing
June 2018

Activity, stability and structure of laccase in betaine based natural deep eutectic solvents.

Int J Biol Macromol 2018 Feb 24;107(Pt B):2574-2579. Epub 2017 Oct 24.

Department of Genetics and Molecular Biology, Medical School, Iran University of Medical Sciences, Tehran, Iran. Electronic address:

Natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms. They are formed by mixing two or more natural compounds in certain ratios producing a liquid having a lower melting point than those of the individual NADES components. Although, the eutectics medium bring several advantages as enhanced solubility of non-polar substrates and/or products, however, these advantages would often be limited by a lower stability of biocatalysts in these systems. To examine this matter, biochemical characterization, thermal stability and tertiary structure of laccase from Bacillus HR03 was investigated as a model in betaine and choline based NADES. In eutectics containing choline, a sudden drop in enzyme activity and stability was observed. Betaine based eutectics exhibited a better media for the laccase stability in comparison with the aqueous buffer and choline chloride eutectics. The enzyme highest activity was observed in 20% (v/v) glycerol:betaine (2:1). Among betaine based eutectics, the enzyme exhibited its highest stability in sorbitol:betaine:water (1:1:1) and glycerol:betaine (2:1) compared to the aqueous buffer at 80 and 90°C. Associated conformational changes caused by solvents were monitored using fluorescence technique. Finally, the effects of NADES on the enzyme activity and stability were discussed.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.10.144DOI Listing
February 2018

Study Break: Glucose-Sensitive Holographic (Bio)Sensors: Fundamentals and Applications

Iran Biomed J 2017 11 15;21(6):347-8. Epub 2017 Aug 15.

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572430PMC
November 2017

Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent.

Appl Microbiol Biotechnol 2017 Oct 11;101(19):7227-7238. Epub 2017 Aug 11.

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K , V, and k values were 5.6 mM, 2.2 μmol/min, and 1.96 × 10 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.
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http://dx.doi.org/10.1007/s00253-017-8456-5DOI Listing
October 2017

Hippocampal asymmetry: differences in the left and right hippocampus proteome in the rat model of temporal lobe epilepsy.

J Proteomics 2017 02 5;154:22-29. Epub 2016 Dec 5.

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

The hippocampus is a complex brain structure and undergoes severe sclerosis and gliosis in temporal lobe epilepsy (TLE) as the most common type of epilepsy. The key features of the TLE may be reported in chronic animal models of epilepsy, such as pilocarpine model. Therefore, the current study was conducted in a rat pilocarpine model of acquired epilepsy. Two-dimensional gel electrophoresis based proteomic technique was used to compare the proteome map of the left and right hippocampus in both control and epileptic rats. Generally, 95 differentially expressed spots out of 1300 spots were identified in the hippocampus proteome using MALDI-TOF-TOF/MS. Within identified proteins, some showed asymmetric expression related to the mechanisms underlying TLE imposed by pilocarpine. Assessment of lateralization at the molecular level demonstrated that expression of proteins involved in dopamine synthesis was significantly more in the right hippocampus than the left one. In the epileptic model, reduction in dopamine pathway proteins was accompanied by an increase in the expression of proteins involved in polyamine synthesis, referring to a new regulating mechanism. Our results revealed changes in the laterality of protein expression due to pilocarpine-induced status epilepticus that could present some new proteins as potential candidates for antiepileptic drug design.

Biological Significance: In the current study, two-dimensional gel electrophoresis (2-DE) based proteomic technique was used to profile changes in the left and right hippocampus proteome after pilocarpine induced status epilepticus. Spots of proteome maps for two hemispheres were excised and identified with MALDI-TOF-TOF/MS. Analysis of proteome map of the left and right hippocampus revealed a lateralization at the molecular level, in which the expression of proteins involved in dopamine synthesis and release were significantly more in right hippocampi than the left ones in the normal rats. Also, the expression of proteins involved in polyamine synthesis significantly increased in epileptic hippocampus (considerably higher in right hippocampi), whilst the proteins which included in dopamine pathways were decreased. Our results revealed changes in the laterality of protein expression due to pilocarpine-induced status epilepticus that could present some new proteins as potential candidates for antiepileptic drug design.
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http://dx.doi.org/10.1016/j.jprot.2016.11.023DOI Listing
February 2017

An alternative allosteric pathway in thermophilic methylglyoxal synthase.

Int J Biol Macromol 2016 Dec 5;93(Pt A):526-533. Epub 2016 Sep 5.

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran 14115-175, Iran. Electronic address:

Methylglyoxal synthase (MGS) is a homohexameric enzyme responsible for converting dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate in the methylglyoxal bypass of glycolysis. Phosphate acts as an allosteric inhibitor and strong regulator for this enzyme. Previous studies on MGS from Thermus sp. GH5 (TMGS) had indicated a pathway for transmitting the signal through Pro82, Arg97 and Val101 to the active site. The necessity of these residues for heterotropic negative cooperativity between subunits of TMGS were also proposed. In this study, it has been shown that a path via a salt bridge between Arg80 and Asp100 in the narrow dimer interface provides an alternative pathway for transmission of the allosteric inhibitory signal through subunit interfaces.
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http://dx.doi.org/10.1016/j.ijbiomac.2016.09.013DOI Listing
December 2016

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

Int J Biol Macromol 2017 Jan 24;94(Pt B):852-858. Epub 2016 Oct 24.

Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran; Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35μg enzyme per 1mg of silicon. The immobilized enzyme displayed lower V values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase.
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http://dx.doi.org/10.1016/j.ijbiomac.2016.10.077DOI Listing
January 2017

Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli.

Protein Expr Purif 2016 10 7;126:42-48. Epub 2016 May 7.

Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran; Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 μM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme's activity without any aggregation.
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http://dx.doi.org/10.1016/j.pep.2016.04.015DOI Listing
October 2016

Development of a label-free SPR sensor for detection of matrixmetalloproteinase-9 by antibody immobilization on carboxymethyldextran chip.

Biosens Bioelectron 2016 Jul 19;81:510-516. Epub 2016 Mar 19.

Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address:

Surface plasmon resonance (SPR) immunosensor has been widely utilized for monitoring antigen-antibody interactions. The sensor measures changes of refractive index upon binding of analyte molecules to specific ligand immobilized on the sensor chip. This effort reports development of SPR immunosensor for real-time and label-free detection of recombinant human matrix metalloproteinases-9 (MMP-9), which has been associated with malignant tumor progression and metastasis by matrix degradation. MMP-9 was expressed in Escherichia coli BL21 and purified by Ni-NTA agarose column. CMD 50 D was activated by EDC/NHS for immobilization of monoclonal anti-MMP-9. Atomic force microscopy images showed uniform distribution of anti-MMP-9 over the sensor chip. Equilibrium constant (KD), maximum binding capacity (Rmax) and ∆Gb values for interaction of MMP-9 and anti-MMP-9 were 0.4nM, 680 µRIU and -53.51kJ/mol, respectively. Concentration of MMP-9 in saliva samples was determined, with linearity in the range of 10-200ng/mL. The limit of detection was found to be 8pg/mL, being lower than most of the previously reported techniques.
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http://dx.doi.org/10.1016/j.bios.2016.03.038DOI Listing
July 2016

A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions.

Int J Biol Macromol 2015 Aug 6;79:822-9. Epub 2015 Jun 6.

Department of Organic Colorants, Institute for Color Science and Technology, Tehran, Iran.

Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher kcat/Km against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π-π and anion-π interactions played the dominant role in stabilizing both variants.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.06.002DOI Listing
August 2015

Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

Biotechnol Appl Biochem 2014 Mar-Apr;61(2):226-9. Epub 2014 Feb 26.

Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.

Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.
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http://dx.doi.org/10.1002/bab.1159DOI Listing
December 2014

Studies on the refolding process of recombinant horseradish peroxidase.

Mol Biotechnol 2013 Jun;54(2):484-92

Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.
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http://dx.doi.org/10.1007/s12033-012-9588-6DOI Listing
June 2013

Molecular detection of human parechovirus type 1 in stool samples from children with diarrhea.

Int J Infect Dis 2012 Sep 4;16(9):e673-6. Epub 2012 Jul 4.

Department of Molecular Biology and Genetics, School of Medicine, Tehran University of Medical Sciences, PO Box 14155-6447, Tehran, Iran.

Background: Human parechovirus type 1 (HPeV-1) appears to be associated with gastrointestinal and respiratory infections, and occasionally with central nervous system symptoms. Since there is no accurate information about the incidence and epidemiology of HPeV-1 in Iran, this study was designed to describe the frequency of HPeV-1 in stool samples from children under 4 years of age with gastroenteritis.

Methods: RNA was isolated from stool suspensions and cDNA was prepared and amplified using a specific nested reverse transcription PCR (RT-PCR). We also compared this RT-PCR method with a cell culture procedure.

Results: Out of 472 samples, 112 (23.7%) were HPeV-1-positive. The incidence and prevalence of this virus was highest in children under 1 year old with diarrhea (p=0.036), and was higher in the spring and autumn than in summer and winter (p<0.001). More males than females were HPeV-1-positive (p<0.001).

Conclusions: This is the first systematic study of the incidence and epidemiology of HPeV-1 among children with acute gastroenteritis in Iran. The results revealed that RT-PCR is a more practical, sensitive, and rapid technique for the detection of HPeV-1 directly from clinical samples than cell culture, which makes this a valuable method for epidemiology. Also, the rapid detection of virus can decrease both the unnecessary use of antibiotics and costs in clinical practice.
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http://dx.doi.org/10.1016/j.ijid.2012.05.1020DOI Listing
September 2012

Extra EF hand unit (DX) mediated stabilization and calcium independency of α-amylase.

Mol Biotechnol 2013 Mar;53(3):270-7

Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.

It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca(2+) per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca(2+) were monitored using fluorescence technique.
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http://dx.doi.org/10.1007/s12033-012-9523-xDOI Listing
March 2013