Tarbiat Modares University


  • Maltogenic amylases (MAases, EC have been gotten much attention due to their various applications in industry and commercial processes. MAases belong to subfamily 20 of glycoside hydrolase ...family 13 (NPase or CDases subfamily) and they have important differences with other members of the family. This enzyme consists of two subunits which form two active sites in the dimer form by binding of the central domain of each subunit to the N domain of the next one (domain-swapping dimeric structure). Allosterism is a possible way of regulating enzymatic activity and no evidence has been found regarding to the cooperativity and correlation between MAases subunits, therefore in this study the allosteric behavior of MAases from a native strain (Geobacillus sp. Gh6) was investigated. Unlike other members of α-amylase family, MAases showed positive cooperativity between their subunits and the enzyme exhibited sigmoidal nature towards all three cyclodextrin (CD) substrates with a Hill constant (nH) value equal to 2, 1.6 and 1.1 for α-CD, β-CD and γ-CD, respectively. On further analysis, the effect of glucose and maltose as MAases allosteric effectors in the presence of β-CD substrate showed that these two effectors had a biphasic effect; while they stimulated the enzyme activity at low concentrations (with a decrease in Hill constant), these metabolites acted as allosteric inhibitors at higher concentrations. Due to the key role of MAases in carbohydrate metabolization, an efficient regulating system for this enzyme is required. In this experiment, for the first time the allosteric properties of MAases were observed and investigated.Show more
    Rahmati P,Sajedi RH,Zamani P,Rahmani H,Khajeh K
    Enzyme and microbial technology
  • Chimeric antigen T cell receptors provide a good approach for adoptive immunotherapy of cancer, especially in the context of cancerous cells that fail to express major histocompatibility complex antig...en and co-stimulatory molecules. Clinical applications of these receptors are limited, mostly due to the xenogenic origin of the antibodies, which cause immunogenic reactions. Nanobodies are the smallest fragments of antibodies that have great homology to human VH and low immunogenic potential. MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in more than 80% of human malignancies. We used anti-MUC1 nanobody as an antigen binding domain, CD28 and CD3zeta as signaling domains, and IgG3 as a spacer in a chimeric receptor construct. This construct was transfected to Jurkat cells. The transfected Jurkat cells were exposed to MUC1-positive MCF7 cells. Then we analyzed the secretion of IL2, proliferation of Jurkat cells, and death of MCF7 cells. These data revealed that the nanobody chimeric receptor can target tumor-associated antigen-positive cells. Regarding the efficient and specific function of nanobody chimeric receptor and non-immunogenic nature of nanobodies, these chimeric receptors might be used as promising candidates for clinical applications.Show more
    Bakhtiari SH,Rahbarizadeh F,Hasannia S,Ahmadvand D,Iri-Sofla FJ,Rasaee MJ
    Hybridoma (2005)
  • Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood gro...up antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH = 8.7, temperature = 14 degrees C, and reaction time = 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.Show more
    Hashemi-Najafabadi S,Vasheghani-Farahani E,Shojaosadati SA,Rasaee MJ,Armstrong JK,Moin M,Pourpak Z
    Bioconjugate chemistry
  • There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used t...o measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.Show more
    Gill P,Forouzandeh M,Rahbarizadeh F,Ramezani R,Rasaee MJ
    Journal of immunoassay & immunochemistry
  • The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, ...eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.Show more
    Zandi K,Roostaee MH,Sadeghizadeh M,Rasaee MJ,Sajedi RH,Soleimanjahi H
    FEMS immunology and medical microbiology
  • Anti-melatonin monoclonal antibodies (MAb) were prepared following coupling melatonin to bovine serum albumin (BSA) by Mannich reaction. Balb/c mice were immunized via injection of the melatonin-BSA i...ntraperitonally. The spleen cells producing high titer of antibody were fused with myeloma cells of SP2/0 origin. After two limiting dilutions, two stable clones (AS-H10 and AS-D26) exhibiting best properties were selected for further studies. The class and subclass of two MAbs were found to be IgG(1) and IgG(2a) with lambda and kappa light chains, respectively. Antibodies secreted by these two clones showed high affinity of about 10(9)M(1). Study of the specificity criteria showed that these clones had no cross reactivity with indolic, aromatic, and imidazole ring-containing compounds, and had high specificity towards melatonin. The calibration curve was constructed with a sensitivity range of 10 ng/mL to 10 microg/mL. In conclusion, these MAbs may be useful for immunoassay of melatonin.Show more
    Soukhtanloo M,Ansari M,Paknejad M,Parizadeh MR,Rasaee MJ
    Hybridoma (2005)
  • PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and syntheti...c peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.Show more
    Mohammadi M,Rasaee MJ,Rajabibazl M,Paknejad M,Zare M,Mohammadzadeh S
    Hybridoma (2005)
  • The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis.
    Pakzad I,Rezaee A,Rasaee MJ,Tabbaraee B,Delpisheh A
    Iranian journal of immunology : IJI
  • Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this st...udy, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.Show more
    Ebrahimi F,Rasaee MJ,Mousavi SL,Babaeipour V
    The Journal of toxicological sciences
  • Worldwide, oral squamous cell carcinoma (potentially mediated by HER2) is recognized as the most commonly occurring malignant neoplasm of the oral cavity. Anti-HER2 nanobodies conjugated to gold-silic...a nanoshells and used as photothermal treatment for oral squamous cell carcinoma may provide a novel therapeutic alternative to current treatment for this disease.Show more
    Fekrazad R,Hakimiha N,Farokhi E,Rasaee MJ,Ardestani MS,Kalhori KA,Sheikholeslami F
    International journal of nanomedicine
  • Aptamers are oligonucleotides with highly structured molecules that can bind to their targets through specific 3-D conformation. Commonly, not all the nucleotides such as primer binding fixed region a...nd some other sequences are vital for aptamers folding and interaction. Elimination of unnecessary regions needs trustworthy prediction tools to reduce experimental efforts and errors. Here we introduced a manipulated in-silico approach to predict the 3-D structure of aptamers and their target interactions. To design an approach for computational analysis of isolated ssDNA aptamers (FLC112, FLC125 and their truncated core region including CRC112 and CRC125), their secondary and tertiary structures were modeled by Mfold and RNA composer respectively. Output PDB files were modified from RNA to DNA in the discovery studio visualizer software. Using ZDOCK server, the aptamer-target interactions were predicted. Finally, the interaction scores were compared with the experimental results. In-silico interaction scores and the experimental outcomes were in the same descending arrangement of FLC112>CRC125>CRC112>FLC125 with similar intensity. The consistent results of innovative in-silico method with experimental outputs, affirmed that the present method may be a reliable approach. Also, it showed that the exact in-silico predictions can be utilized as a credible reference to find aptameric fragments binding potency.Show more
    Heiat M,Najafi A,Ranjbar R,Latifi AM,Rasaee MJ
    Journal of biotechnology
  • Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of ant...ibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.Show more
    Khalili E,Lakzaei M,Rasaee MJ,Aminian M
    Monoclonal antibodies in immunodiagnosis and immunotherapy
  • Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were deter...mined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.Show more
    Rezaee MA,Rasaee MJ,Mohammadnejad J
    Journal of immunoassay & immunochemistry
  • PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to compare the two labeling methods (direct and... indirect radioiodination) for application of this antibody against MUC1 as a radioimmunotherapeutical agent.Monoclonal antibody (PR81) against the tandem repeat of the core protein (MUC1) was prepared, characterized, purified, and labeled with 131I using the direct (chloramin-T) and indirect (Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH (YYK) attached to N-hydroxysuccinimide as a linker between PR81 and 131I) methods. The immunoreactivity of 131I-PR81 and 131I-TP-PR81 complexes with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of 131I-PR81 and 131I-YYK-peptide-PR81 complexes in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the 131I-PR81 and 131I- YYK-peptide -PR81 complexes was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The labeling efficiency was determined by measuring the percentage recovery of radioactivity in the final product relative to the initial activity in the shipment vial, was found to be 59.9% +/- 7.9% for direct and 50% +/- 3.2% for indirect methods. 131I-PR81 and 131I- YYK- peptide -PR81 complexes showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled products in human serum which was measured by thin layer chromatography (TLC) was found to be more than 50% over 24 hr for 131I-PR81 and 70% for 131I- YYK-peptide -PR81 complexes. Cell toxicity and in vitro internalization studies showed that the 131I-PR81 and 131I- YYK-peptide -PR81 complexes inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to 40% of the 131I-PR81 and 60% of the 131I- YYK-peptide -PR81 complexes internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection. Thyroid and stomach levels from PR81 labeled with 131I- YYK-peptide were two- to three- fold less than those with directly labeled 131I-PR81, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinase. These results show that the indirect labeling was better than the indirect labeling and 131I- YYK-peptide -PR81 may be considered as a promising candidate for therapy of breast cancer.Show more
    Mohammadnejad J,Rasaee MJ,Babaei MH,Paknejad M,Hasan ZM,Salouti M,Gandomkar M,Sadri K
    Human antibodies
  • PR81 is a monoclonal antibody that binds with high affinity to MUC1 antigen that is over expressed in 80% of breast cancers. In this study, we developed a method for indirect labeling of PR81 with lut...etium-177 and performed all preclinical qualifications in production of a biologic agent for radioimmunotherapy of breast cancer.Show more
    Salouti M,Babaei MH,Rajabi H,Rasaee Mj
    Nuclear medicine and biology
  • We examined the effect of exercise intensity and mode on the acute responses of free testosterone to cortisol ratio and salivary α-amylase. We also evaluated the relationship between cortisol and sali...vary α-amylase. Ten healthy young active males participated voluntarily in this study in six single sessions. They exercised on a cycle ergo meter, treadmill, and elliptical instrument at intensities of 70% and 85% maximum heart rate for 25 minutes. Saliva samples were collected 5 minutes before and 5 minutes after each exercise session. No significant changes were observed for cortisol. Free testosterone to cortisol ratio increased during each exercise session (F5, 45=3.15, P=0.02). However, these changes are only significant after exercise on the treadmill at 70% maximum heart rate (t=2.94, P=0.02) and 85% maximum heart rate (t=0.53, P=0.03). Salivary α-amylase significantly varied among exercise sessions (F5, 45=3.97, P=0.005), and a significant decline was observed after exercise on the elliptical instrument (t=2.38, P=0.04) and treadmill (t=3.55, P=0.006) at 85% maximum heart rate. We found that the free testosterone to cortisol ratio is dependent on the exercise mode, while the salivary α-amylase response is dependent on the intensity of exercise. The increase of free testosterone to cortisol ratio in this study may indicate lower physiological stress in response to performing these exercises. Applying muscular strength with moderate intensity weight-bearing exercises possibly activates the anabolic pathways. Although the cortisol and salivary α-amylase responses were opposite in the majority of the exercise sessions, no significant inverse relationship was observed.Show more
    Azarbayjani MA,Fatolahi H,Rasaee MJ,Peeri M,Babaei R
    International journal of exercise science
  • The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been us...ed to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.Show more
    Iri-Sofla FJ,Rahbarizadeh F,Ahmadvand D,Rasaee MJ
    Experimental cell research
  • This study examined the effects of pre season training on restring level and acute response of mucosal immunity in male basketball players.
    Azarbayjani M,Nikbakht H,Rasaee MJ
    The Journal of sports medicine and physical fitness
  • We digested anti-MUC1 monoclonal antibody PR81 to produce F(ab)₂ fragments. A comparison was performed between the two radiolabeled PR81 and F(ab)₂ fragments for breast tumor imaging in a mouse model.
    Salouti M,Babaei MH,Rajabi H,Foroutan H,Rasaee MJ,Rajabi AB,Mohammadnejad J,Shafiee M,Mazidi M,Daha FJ
    Annals of nuclear medicine
  • In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-st...imulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.Show more
    Khaleghi S,Rahbarizadeh F,Ahmadvand D,Rasaee MJ,Pognonec P
    International journal of hematology
  • A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. ...Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.Show more
    Dehghannezhad A,Paknejad M,Rasaee MJ,Omidfar K,Seyyed Ebrahimi SS,Ghahremani H
    Hybridoma (2005)
  • We provide evidence for combining a single domain antibody (nanobody)-based targeting approach with transcriptional targeting as a safe way to deliver lethal transgenes to MUC1 over-expressing cancer ...cells. From a nanobody immune library, we have isolated an anti-DF3/Mucin1 (MUC1) nanobody with high specificity for the MUC1 antigen, which is an aberrantly glycosylated glycoprotein over-expressed in tumours of epithelial origin. The anti-MUC1 nanobody was covalently linked to the distal end of poly(ethylene glycol)(3500) (PEG(3500)) in PEG(3500)-25kDa polyethylenimine (PEI) conjugates and the resultant macromolecular entity successfully condensed plasmids coding a transcriptionally targeted truncated-Bid (tBid) killer gene under the control of the cancer-specific MUC1 promoter. The engineered polyplexes exhibited favourable physicochemical characteristics for transfection and dramatically elevated the level of Bid/tBid expression in both MUC1 over-expressing caspase 3-deficient (MCF7 cells) and caspase 3-positive (T47D and SKBR3) tumour cell lines and, concomitantly, induced considerable cell death. Neither transgene expression nor cell death occurred when the MUC1 promoter was replaced with the CNS-specific synapsin I promoter. Since PEGylated PEI was only responsible for DNA compaction and played no significant role in direct transfection and cell killing, our attempts overcome previously reported PEI-mediated apoptotic and necrotic cell death, which is advantageous for future in vivo transcriptional targeting as this will minimize (or eliminate) non-targeted cell damage.Show more
    Sadeqzadeh E,Rahbarizadeh F,Ahmadvand D,Rasaee MJ,Parhamifar L,Moghimi SM
    Journal of controlled release : official journal of the Controlled Release Society
  • With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin... (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.Show more
    Kouchakzadeh H,Shojaosadati SA,Mohammadnejad J,Paknejad M,Rasaee MJ
    Human antibodies
  • The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of tre...ated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).Show more
    Alirezapour B,Rajabibazl M,Rasaee MJ,Omidfar K
    Monoclonal antibodies in immunodiagnosis and immunotherapy
  • Single domain antibodies from camel heavy chain antibodies (VHH or nanobody), are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecu...lar weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals.Show more
    Rajabibazl M,Rasaee MJ,Forouzandeh M,Rahimpour A
    Iranian journal of immunology : IJI
  • The direct effect of some opioids on immune cells has been demonstrated. The aim of this study was to assess the apoptotic effect of opium on Jurkat T lymphocyte cells.
    Igder S,Asadikaram GR,Sheykholeslam F,Sayadi AR,Mahmoodi M,Kazemi Arababadi M,Rasaee MJ
    Addiction & health
  • Due to the high atomic number of gold nanoparticles (GNPs), they are known as new radiosensitizer agents for enhancing the efficiency of superficial radiotherapy techniques by increasing the dose abso...rbed in tumor cells wherein they can be accumulated selectively. The aim of this study was to compare the effect of various common low energy levels of orthovoltage x-rays and megavoltage γ-rays (Co-60) on enhancing the therapeutic efficiency of HeLa cancer cells in the presence of conjugated folate and non-conjugated (pegylated) GNPs. To achieve this, GNPs with an average diameter of 52 nm were synthesized and conjugated to folic acid molecules. Pegylated GNPs with an average diameter of 47 nm were also synthesized and used as non-conjugated folate GNPs. Cytotoxicity assay of the synthesized folate-conjugated and pegylated GNPs was performed using different levels of nanoparticle concentration incubated with HeLa cells for 24 h. The radiosensitizing effect of both the conjugated and pegylated GNPs on the cells at a concentration of 50 µM was compared using MTT as well as clonogenic assays after exposing them to 2 Gy ionizing radiation produced by an orthovoltage x-ray machine at four different kVps and γ-rays of a Co-60 unit. Significant differences were noted among various irradiated groups with and without the folate conjugation, with an average dose enhancement factor (DEF) of 1.64 ± 0.05 and 1.35 ± 0.05 for the folate-conjugated and pegylated GNPs, respectively. The maximum DEF was obtained with the 180 kVp x-ray beam for both of the GNPs. Folate-conjugated GNPs can significantly enhance the cell killing potential of orthovoltage x-ray energies (especially at 180 kVp) in folate receptor-expressing cancer cells, such as HeLa, in superficial radiotherapy techniques.Show more
    Khoshgard K,Hashemi B,Arbabi A,Rasaee MJ,Soleimani M
    Physics in medicine and biology
  • Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced prote...in during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.Show more
    Mohammadzadeh S,Rajabibazl M,Fourozandeh M,Rasaee MJ,Rahbarizadeh F,Mohammadi M
    Monoclonal antibodies in immunodiagnosis and immunotherapy
  • To date, several small molecule inhibitors and monoclonal-antibodies (like ICR-62) have been used to treat tumors over-expressing epidermal growth factor receptor (EGFR). However, the limitations asso...ciated with these conventional applications accentuate the necessity of alternative approaches. Mimotopes as compelling molecular tools could rationally be employed to circumvent these drawbacks. In the present study, an M13 phage displaying ICR-62 binding peptide mimotope is exploited as a vaccine candidate. It exhibited high affinity towards ICR62 and polyclonal anti-P-BSA antibodies. Following the mice immunization, phage-based mimotope vaccine induced humoral immunity. Elicited anti-EGFR mimotope antibodies were detected using ELISA method. Moreover, the phage vaccine was tested on the Lewis lung carcinoma mice model to investigate the prophylactic and therapeutic effects. The tumor volume was measured and recorded in different animal groups to evaluate the anti-tumor effects of the vaccine. Our data indicate that the reported phage-based mimotope could potentially elicit specific antibodies resulting in low titers of EGFR-specific antibodies and reduced tumor growth. However, in vivo experiments of prophylactic or therapeutic vaccination showed no specific advantage. Furthermore, phage-mimotope vaccine might be a promising approach in the field of cancer immunotherapy.Show more
    Asadi-Ghalehni M,Ghaemmaghami M,Klimka A,Javanmardi M,Navari M,Rasaee MJ
    Immunopharmacology and immunotoxicology
  • Individual preparation of two human T-cell lymphotropic virus type I (HTLV-I) diagnostic GST fused peptides (MTA-1 and GD21) is time-consuming and expensive. The aim of this study was to design a nove...l single chimeric antigen (SCA) to obviate separate expression of proteins and reduce the cost of reagent preparation. Structural protein fragments, including immunodominant B cell linear epitopes, were selected and different SCAs were designed. Tertiary structure, epitope exposure, solubility and stability were calculated for each SCA and compared with each other. The synthetic DNA encoding the interested SCA was sub-cloned into pET32a expression vector, expressed as a soluble form in Escherichia coli BL21 (DE3) cells and purified under native condition using affinity chromatography. The SDS-PAGE results indicated that thioredoxin-fused SCA was successfully expressed as a soluble form in E. coli BL21 (DE3) cells. The results of ELISA confirmed that SCA reacted with anti-HTLV-I antibodies in a concentration-dependent manner. Our results indicated that the designed SCA may be a good candidate for the screening of HTLV-I carriers with antigen-antibody-based tests.Show more
    Heydari Zarnagh H,Hassanpour K,Rasaee MJ
    Iranian journal of allergy, asthma, and immunology
  • One of the proposed approaches in cancer therapy is to induce and direct the patients own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epi...dermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.Show more
    Navari M,Zare M,Javanmardi M,Asadi-Ghalehni M,Modjtahedi H,Rasaee MJ
    Immunopharmacology and immunotoxicology
  • Breast cancer radioimmunoscintigraphy targeting MUC1 expression is a growing field of work in nuclear medicine research. PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is o...ver expressed on breast tumors. In this study, we report production, quality control and preclinical qualifications of a copper-64 labeled PR81 for PET imaging of breast cancer.Show more
    Alirezapour B,Rasaee MJ,Jalilian AR,Rajabifar S,Mohammadnejad J,Paknejad M,Maadi E,Moradkhani S
    Nuclear medicine and biology
  • Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of ...anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3) cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.Show more
    Heydari Zarnagh H,Ravanshad M,Pourfatollah AA,Rasaee MJ
    Iranian journal of allergy, asthma, and immunology
  • Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the ...high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.Show more
    Heiat M,Ranjbar R,Latifi AM,Rasaee MJ
  • Nucleic acid aptamers can be served as drugs, carriers and diagnostic probes in living systems. Before recruiting aptamers, their pharmacological characteristics should be determined. Here we intended... to investigate four important properties of isolated ssDNA anti-angiotensin II aptamers (FLC112 and FLC125) including hemolytic activity, cytotoxicity, immunogenicity and serum stability through in vitro and in vivo models. The hemolytic effect and cytotoxicity potential of aptamers were measured through hemolysis test and MTT assay respectively. In the following test, the humoral immune responses to aptamers in BALB/c mice were assessed. The human serum stability of aptamers was also determined using real-time PCR (qPCR). The results of this study revealed that the FLC112 aptamer with its unique structure had slightly higher cytotoxicity and hemolysis effect (9.14% and 0.1 ± 0.037% respectively) relative to FLC125 (8.07% and 0.08 ± 0.045% respectively) at the highest concentration (5 μM). FLC112 showed ignorable immune response in mice and barely higher than FLC125. Serum stability test confirmed that FLC112 with 12 h had more nuclease stability than FLC125 with 8 h. Aptamer molecule analysis revealed that the structure, sequense composition and motifs are the determinative parameters in aptamer pharmacological properties.Show more
    Heiat M,Ranjbar R,Fasihi-Ramandi M,Latifi AM,Rasaee MJ
    Molecular and cellular probes
  • Designing novel antigens to rise specific antibodies for Thyroid Stimulating Hormone (TSH) detection is of great significance. A novel fusion protein consisting of the C termini sequence of TSH beta s...ubunit and a fusion sequence was designed and produced for rabbit immunization. Thereafter, the produced antibodies were purified and characterized for TSH detection. Our results indicate that the produced antibody is capable of sensitive and specific detection of TSH with low cross reactivity. This study underscores the applicability of designed fusion protein for specific and sensitive polyclonal antibody production and the importance of selecting an amenable region of the TSH for immunization.Show more
    Mard-Soltani M,Rasaee MJ,Sheikhi A,Hedayati M
    Journal of immunoassay & immunochemistry
  • Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient depriva...tion in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-β-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.Show more
    Aghamollaei H,Mousavi Gargari SL,Ghanei M,Rasaee MJ,Amani J,Bakherad H,Farnoosh G
    Biotechnology and applied biochemistry
  • Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with its significance in a var...iety of biological processes accentuate the necessity to decipher its 3D structure that would pave the way towards the development of relevant selective inhibitors. A DKK1 structure model predicted by the Robetta server with structural refinements including a 10 ns molecular dynamics run was subjected to functional and docking analyses. We hypothesize that the N-terminal region of the DKK1 molecule could be functionally important for both canonical and noncanonical Wnt pathways. Moreover, it seems that DKK1 could be involved in interactions with the Frizzled receptors, leading to the activation of the Planar Cell Polarity (PCP) pathway (activation of Jun N-terminal kinase (JNK) Pathway) and Wnt/Ca^(2+) pathway (activation of CamKII).Show more
    Khalili S,Rasaee MJ,Bamdad T
    Molekuliarnaia biologiia
  • Asymmetric PCR, a simple method to generate single-stranded DNA (ssDNA) aptamers in systematic evaluation of ligand by exponential enrichments rounds, is coupled with limitations. We investigated the ...essential strategies for optimization of conditions to perform a high-quality asymmetric PCR. Final concentrations of primers and template, the number of PCR cycles, and annealing temperature were selected as optimizing variables. The qualities of visualized PCR products were analyzed by ImageJ software. The highest proportion of interested DNA than unwanted products was considered as optimum conditions. Results revealed that the best values for primers ratio, final template concentration, annealing temperature, and PCR cycles were, respectively, 30:1, 1 ng/μL, 55 °C, and 20 cycles for the first and 50:1, 2 ng/μL, 59 °C, and 20 cycles for other rounds. No significant difference was found between optimized asymmetric PCR results in the rounds of two to eight (P > 0.05). The ssDNA quality in round 10 was significantly better than other rounds (P < 0.05). Generally, the ssDNA product with less dimers, double-stranded DNA (dsDNA), and smear are preferable. The dsDNA contamination is the worst, because it can act as antidote and inhibits aptameric performance. Therefore, to choose the best conditions, the lower amount of dsDNA is more important than other unwanted products.Show more
    Heiat M,Ranjbar R,Latifi AM,Rasaee MJ,Farnoosh G
    Biotechnology and applied biochemistry
  • Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the EGF receptor in many human tumors. This variant is tumor specific and highly immunogenic, thus, it can be used... as a target for targeted drug delivery toward tumor cells. The major aim of this study was to develop an EGFRvIII-mediated drug delivery system by anti-EGFRvIII monoclonal antibody (MAb) conjugated to doxorubicin (Dox)-loaded nanostructured lipid carriers (NLC) to enhance the targeting specificity and cytotoxic effect of Dox on EGFRvIII-overexpressing cell line. In our study, Dox was chosen as a hydrophobic cytotoxic drug and drug-loaded nanostructured lipid carriers (Dox-NLC) was prepared by solvent emulsification/evaporation method. In order to conjugate anti-EGFRvIII MAb to Dox-NLC, DSPE-PEG2000-NHS (1,2-distearoylphosphatidylethanolamine-polyethylene glycol 2000-NHS) was used as a linker. Physicochemical characteristics of antibody conjugated Dox-NLC (MAb-Dox-NLC), including particle size, zeta potential, entrapment efficiency and in vitro Dox release were investigated. Cytotoxicity of MAb-Dox-NLC against NIH-3T3 and HC2 20d2/c (EGFRvIII-transfected NIH-3T3) cell lines was evaluated. The MAb-Dox-NLC appeared to enhance the cytotoxic activity of targeted NLC against HC2 20d2/c cells. The cellular uptake percentage of targeted NLC by HC2 20d2/c cells was higher than that of NIH-3T3 cells, indicating that EGFRvIII can specifically target HC2 20d2/c cells. In conclusion, anti-EGFRvIII MAb-targeted NLC may be considered as an effective nanocarrier for targeted drug delivery.Show more
    Abdolahpour S,Toliyat T,Omidfar K,Modjtahedi H,Wong AJ,Rasaee MJ,Kashanian S,Paknejad M
    Artificial cells, nanomedicine, and biotechnology
  • A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicill...inase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.Show more
    Rassaie MJ,Kumari LG,Pandey PK,Gupta N,Kochupillai N,Grover PK
  • Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hy...droxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.Show more
    Rassaie MJ,Kumari GL,Rao PN,Shrivastav TG,Pandey HP
  • Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wid...e variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml.Show more
    Dorraj GS,Rassaee MJ,Latifi AM,Pishgoo B,Tavallaei M
    Journal of biotechnology
  • Mnemiopsin, an EF-hand Ca(2+) binding photoprotein isolated from luminous ctenophore Mnemiopsis leidyi, emits blue light from its chromophore, coelenterazine, which is non-covalently bond in its centr...al hydrophobic core. Previous studies have revealed unique biochemical properties for ctenophore photoproteins such as inactivation by light, but only few have focused on photoinactivation process. To understand the nature of photoinactivation process we have investigated the impact of light alone and in the presence of Ca(2+) ion on the structure of this photoprotein. We used UV-Vis, circular dichroism (CD) and fluorescence spectroscopy following Ca(2+) binding assay to analyze the light effects on mnemiopsin conformation in comparison with aequorin at both apo and holo form. Our results showed light induced structural changes which resulted into photoinactivation. These changes include significant modification on secondary structure of mnemiopsin in comparison with aequorin. Our data also revealed that light could influence structure of apo protein regardless of presence of coelenterazine. The comparative studies of Ca(2+) ion binding affinity following light exposure, also showed that light induced structural changes could presumably affect coelenterazine binding or its conformation in binding site in such a way that causes photoinactivation. In conclusion, we have proposed that structural rearrangement of helix 5 and C-terminal motif could be responsible for light induced structural changes.Show more
    Pashandi Z,Molakarimi M,Sajedi RH,Taghdir M,Naderi-Manesh H
    Journal of photochemistry and photobiology. B, Biology
  • A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence ide...ntity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl(2). The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.Show more
    Aghamaali MR,Jafarian V,Sariri R,Molakarimi M,Rasti B,Taghdir M,Sajedi RH,Hosseinkhani S
    The protein journal
  • Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g ...aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovins structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.Show more
    Pashandi Z,Molakarimi M,Mohseni A,Sajedi RH,Taghdir M,Naderi-Manesh H
    Biochemical and biophysical research communications
  • A number of complex and rare mutations in epidermal growth factor receptor (EGFR) gene have been identified and the clinical implication of serum EGFR ligands has also been reported. However, the prog...nostic significance of these mutations and also the serum EGFR and its ligands in Non-Small Cell Lung Carcinoma (NSCLC) has remained a challenging issue. This study is aimed at finding the prognostic importance of EGFR rare mutations and serum EGFR, amphiregulin (AR), and TGF-α (Transforming Growth Factor-alpha) in NSCLC.Show more
    Haghgoo SM,Khosravi A,Mortaz E,Pourabdollah-Toutkaboni M,Seifi S,Sabour S,Allameh A
    Clinical biochemistry
  • This study was performed in response to the rapid propagation of HIV/AIDS across Iran and its status in this region. Accordingly, an evidence-based program is required to combat this disease.
    Haghgoo SM,Joula H,Mohammadzadeh R,Sabour S,Yousefi R,Ghahramani G,Rahimi AA
    Jundishapur journal of microbiology
  • Recent studies have been established high degree of genetic diversity in solid organ tumors among individuals and even between individual tumor cells. This intratumor and intertumor genetic diversity ...results in a heterogeneous tumor with unique characteristics which potentially allows effective drug therapy. The goal of pharmacogenomics is to elucidate the genetic network(s) that underlie drug efficacy and drug resistance. Advances in targeted and personalized therapy play an increasingly important role in many common cancers, notably lung cancer, due to the high incidence, prevalence, mortality and the greater tendency towards drug resistance seen in these patients. Non-small cell lung cancer (NSCLC) is characterized by mutations in the epidermal growth factor receptor (EGFR) and or downstream kinase pathways. This has led to the development of highly selective monoclonal antibodies and EGFR tyrosine kinase inhibitors (EGFR-TKIs) to prevent cancer initiation, proliferation, differentiation, angiogenesis, survival, and invasion. However, resistance to many of these new treatments is induced and further pharmacogenomic analysis has revealed mutations associated with increased or reduced drug efficacy. Combinations of kinase inhibitors or potentially the targeting of cancer stem cells may further increase the success of pharmacogenomics in treating patients with lung cancer.Show more
    Haghgoo SM,Allameh A,Mortaz E,Garssen J,Folkerts G,Barnes PJ,Adcock IM
    European journal of pharmacology
  • Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in thi...s crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H2O2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions.Show more
    Takalloo Z,Sajedi RH,Hosseinkhani S,Moazzenzade T
    International journal of biological macromolecules
  • Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was pr...eviously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones.Show more
    Takalloo Z,Sajedi RH,Hosseinkhani S,Asghari SM
    Archives of biochemistry and biophysics
  • As a Ca(2+)-regulated photoprotein, aequorin (Aeq) contains four EF-hand motifs, the second one lacks the standard sequence for Ca(2+) coordination and doesnt bind to Ca(2+). Here, we replaced this lo...op with a functional loop. According to structural studies, although the global stability of modified aequorin (4EFAeq) is higher than that of Aeq; increasing the local flexibility accompanied by internal structural rearrangements in 4EFAeq result in its penetrability to urea and acrylamide. A fast decay rate was observed for 4EFAeq. Assuming the presence of intermediate states in the luminescent reaction, this observation indicate that the loop replacement leads to the lowering of the half-life of intermediate states which results in increasing the rate of conformational switching of 4EFAeq to light emitting form. However, considerable reduction in initial luminescence intensity of 4EFAeq suggests that the number of functional complexes is reduced. Our findings demonstrate that the conformational effects of the second loop in Aeq elicit a delicate balance between local flexibility and global stability which may be considered as an important functional parameter in photoproteins. It was also concluded that evolutionary conservation of EF-hand ΙΙ in the current form is a consequence of priority of intensity to decay rate in bioluminescent organisms.Show more
    Ebrahimi M,Mohseni A,Khalifeh K,Ranjbar B,Sajedi RH
    Archives of biochemistry and biophysics
  • Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g ...aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovins structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.Show more
    Pashandi Z,Molakarimi M,Mohseni A,Sajedi RH,Taghdir M,Naderi-Manesh H
    Biochemical and biophysical research communications
  • Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arran...gement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.Show more
    Molakarimi M,Mohseni A,Taghdir M,Pashandi Z,Gorman MA,Parker MW,Naderi-Manesh H,Sajedi RH
    PloS one