An emphatic study on the luciferin-luciferase bioluminescence system of Benthosema pterotum.

Approximately 80% of luminous organisms live in the oceans, and considerable diversity of life dependence on bioluminescence has been observed in marine organisms. Among vertebrates, luminous fish species are the only group of vertebrates that have the ability to emit bioluminescent light. Meanwhile, the lantern fish family (Myctophidae), with 33 genera all of which have the ability to emit light, is considered the most prominent family among the luminous fish of the deep oceans and seas. Lantern fish Benthosema pterotum has bioluminescence properties due to the presence of photophores scattered in its ventral-lateral region. However, no research has been performed on its bioluminescence system and light emission mechanism. The present research aimed to assess the type of bioluminescence, pigment, photoprotein, or luciferin-luciferase system in B. pterotum. In order to determine the type of light-emitting system in B. pterotum species, several specific experiments were designed and performed. It was shown that the light emission system in B. pterotum species is categorized into the luciferin-luciferase type. Conducting this research was not only innovative, but it also could be the beginning of further research in the field of marine biochemistry and production of the recombinant active forms of enzymes for industrial, commercial, medical, and pharmaceutical purposes.

Rapid and simple screening of the apoptotic compounds based on Hsp70 inhibition using luciferase as an intracellular reporter.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.

ECM-Dependence of Endothelial Progenitor Cell Features.

Preserving self-renewal, multipotent capacity, and large-scale expansion of highly functional progenitor cells, including endothelial progenitor cells (EPCs), is a controversial issue. These current limitations, therefore, raise the need of developing promising in vitro conditions for prolonged expansion of EPCs without loss of their stemness feature. In the current study, the possible role of three different natural extracellular substrates, including collagen, gelatin, and fibronectin, on multiple parameters of EPCs such as cell morphology, phenotype, clonogenic, and vasculogenic properties was scrutinized. Next, EPCs from GFP-positive mice were pre-expanded on each of these ECM substrates and then systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed considerable promise for fibronectin for EPC expansion with maintenance of stemness characteristics, whereas gelatin and collagen matrices directed the cells toward a mature endothelial phenotype. Transplantation of EPCs pre-expanded on fibronectin resulted in widespread distribution and appropriate engraftment to various tissues with habitation in close association with the microvasculature. In addition, fibronectin pre-expanded cells were gradually enriched in the bone marrow after transplantation, resulting in marrow repopulation and hematologic recovery, leading to improved survival of recipient mice whereas gelatin- and collagen-expanded cells failed to reconstitute the bone marrow. This study demonstrated that, cell characteristics of in vitro expanded EPCs are determined by the subjacent matrix. Fibronectin-expanded EPCs are heralded as a source of great promise for bone marrow reconstitution and neo-angiogenesis in therapeutic bone marrow transplantation. J. Cell. Biochem. 117: 1934-1946, 2016. © 2016 Wiley Periodicals, Inc.

Human serum miR-34a as an indicator of exposure to ionizing radiation.

Radiation exposure in industrial accidents or nuclear device attacks is a major public health concern. There is an urgent need for markers that rapidly identify people exposed to ionizing radiation (IR). Finding a blood-based marker is advantageous because of the ease of sample collection. This study was designed to test the hypothesis that serum miR-34a could serve as an indicator of exposure to IR. Therefore, 44 women with breast cancer, where radiotherapy was part of their therapeutic protocol, were investigated in this study. After demonstrating the appropriateness of our microRNA (miRNA) extraction efficiency and miRNA assay in human serum, we analyzed the miR-34a level in paired serum samples before and after radiotherapy. Fifty Gy X-ray irradiation in daily dose fractions of 2 Gy, 5 days per week, was used in this study. We demonstrated that IR significantly increased serum level of miR-34a. By measuring miR-34a in serum, we could distinguish irradiated patients with sensitivity of 65 % and specificity of 75 %. According to this study, serum miR-34a has the potential to be used as an indicator of radiation exposure.

Effect of exercise training on saliva brain derived neurotrophic factor, catalase and vitamin c.

Background: The balance between production of Reactive Oxygen Species (ROS) and antioxidant defense in the body has important health implications. The aim of this study was to investigate the changes in salivary antioxidants: catalase, vitamin C and brain-derived neurotrophic factor (BDNF), in sedentary men at rest and after acute exhaustive exercise. Methods: This randomized controlled clinical trial (The registry code IRCT2011053212431N1) recruited twenty-five sedentary men (age=21±3yrs; height=172±8cm; weight=66±9kg; VO2 max=37.6±7.4mL•kgkg-1•min-1) participated in a double-blind randomized experiment. Unstimulated whole saliva samples were collected before, immediately and 1 hour after exhaustive treadmill running. Catalase, vitamin C (Vit C) concentration, and BDNF concentrations were determined using biochemical assays and ELISA respectively. Repeated measures ANOVA and Bonferroni posthoc test were used to analyze data. Results: The results of the present study showed that an acute intensive exercise causes a reduction in salivary catalase, Vit C and also BDNF concentration (p<0.05) compared with pre-exercise. Both catalase and Vit C showed a tendency to return to pre-exercise value after one hour. However, BDNF continued to reduction at least 1 hour after the ending of the training. Conclusion: Reduction in antioxidants capacity of saliva might reflects disturbance in natural antioxidant defense mechanisms of the body after an acute intensive physical stress and possible further health threatening consequences.

Breast cancer photothermal therapy based on gold nanorods targeted by covalently-coupled bombesin peptide.

Photothermal therapy, a minimally invasive treatment method for killing cancers cells, has generated a great deal of interest. In an effort to improve treatment efficacy and reduce side effects, better targeting of photoabsorbers to tumors has become a new concept in the battle against cancer. In this study, a bombesin (BBN) analog that can bind to all gastrin-releasing peptide (GRP) receptor subtypes was bound covalently with gold nanorods (GNRs) using Nanothinks acid as a link. The BBN analog was also coated with poly(ethylene glycol) to increase its stability and biocompatibility. The interactions were confirmed by ultraviolet-visible and Fourier transform infrared spectroscopy. A methylthiazol tetrazolium assay showed no cytotoxicity of the PEGylated GNR-BBN conjugate. The cell binding and internalization studies showed high specificity and uptake of the GNR-BBN-PEG conjugate toward breast cancer cells of the T47D cell line. The in vitro study revealed destruction of the T47D cells exposed to the new photothermal agent combined with continuous-wave near-infrared laser irradiation. The biodistribution study showed significant accumulation of the conjugate in the tumor tissue of mice with breast cancer. The in vivo photothermal therapy showed the complete disappearance of xenographted breast tumors in the mouse model.

Immobilized papain on gold nanorods as heterogeneous biocatalysts.

Papain, a thiol protease present in the latex of Carica papaya, is an enzyme which exhibits broad proteolytic activity, and, for this reason, it is utilized in a variety of industrial applications. Immobilization of papain on gold nanoparticles highly preserves its activity and enhances the stability, allowing the reuse of the linked enzyme many times without any significant loss of its catalytic performance. In particular, kcat and KM values remain substantially unchanged, while immobilized form shows a higher activity on a wider pH range retains 80 % residual activity also at 90 °C and shows higher functionality than the free form when incubated for long time (1 h) at 90 °C and at extreme pH values (3 and 12). A higher activity of immobilized papain with respect to the free form in the presence of various bivalent metal ions, known as strong inhibitors of papain, was also found. The reasons of this enhanced stability of gold nanorods immobilized papain are discussed.

Uncovering the role of sorbitol in Renilla luciferase kinetics: Insights from spectroscopic and molecular dynamics studies.

Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide, resulting in the emission of a photon of light. This study investigated the impact of sorbitol on the structural and kinetic properties of Renilla luciferase using circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. Our investigation, carried out using circular dichroism and fluorescence analyses, as well as a thermal stability assay, has revealed that sorbitol induces conformational changes in the enzyme but does not improve its thermal stability. Moreover, through kinetic studies, it has been demonstrated that at a concentration of 0.4 M, sorbitol enhances the catalytic efficiency of Renilla luciferase. However, at higher concentrations, sorbitol results in a decrease in catalytic efficiency. Additionally, molecular dynamics simulations have shown that sorbitol increases the presence of hydrophobic pockets on the enzyme's surface. These simulations have also provided evidence that at a concentration of 0.4 M, sorbitol facilitates substrate access to the active site of the enzyme. Nevertheless, at higher concentrations, sorbitol obstructs substrate trafficking, most likely due to its impact on the gateway to the active site. This study may provide insights into the kinetic changes observed in enzymes with buried active sites, such as those with α/ß hydrolase fold.

Site-directed mutagenesis of photoprotein mnemiopsin: implication of some conserved residues in bioluminescence properties.

Mnemiopsin is a Ca(2+)-binding photoprotein from Mnemiopsis leidyi that emits a flash of blue light upon reacting with coelenterazine and Ca(2+). The light emission is a result of an intramolecular oxidation reaction. Similar to the other Ca(2+)-binding photoproteins, mnemiopsin is composed of apophotoprotein (206 amino acid residues), the imidazopyrazine chromophore, coelenterazine, and molecular oxygen. The biochemical properties of this photoprotein have been recently characterized but so far there has been no individual study on the role of critical residues. In this study, we introduced some mutations in the mnemiopsin structure for investigation of the roles of some critical residues in the substrate binding cavity, and neighboring residues in the mechanism of the reaction and the bioluminescence properties of the photoprotein. Mutants of mnemiopsin were produced by substitution of residues M77, W101 and M151. Three mutants (W101F, W101Y and M151Y mutants) had significantly reduced luminescence activity and altered bioluminescent properties (such as decay rate, Ca(2+) sensitivity, etc.), whereas the fourth (M77H mutant) lost its luminescence activity completely. Our experimental and theoretical studies suggest that residue M77 probably has structural importance and participates in stabilization of active site residues, whereas residue M151 is one of the critical mechanistic residues in ctenophore photoproteins.

Polyphyllin D-loaded solid lipid nanoparticles for breast cancer: Synthesis, characterization, in vitro, and in vivo studies.

Polyphyllin D (PD), a steroidal saponin in Paris polyphylla, induces apoptosis via the intrinsic apoptotic pathway in different cancer types. However, emerging evidence has shown that the primary issue with PD is its structure's hemolysis and cytotoxicity. This study aimed to develop and optimize PD-loaded SLN formulation and evaluate its efficacy in breast cancer cell lines. Apoptosis, as the mechanism of cell death, was confirmed by flow cytometry following Annexin V/propidium iodide staining and western blot analysis. In in vivo studies, tumor inhibitory efficacy was compared with different doses of PD-loaded SLN on 4T1-implanted BALB/c mice. The half-maximal inhibitory concentration (IC50) of PD- loaded SLN was calculated to be 33.25 and 35.74 µg/mL for MCF7 and MDA-MB-231 cells, respectively. Flow cytometry analysis further confirmed a significant increase in apoptosis after treatment with PD- loaded SLN. When both cell lines were treated with PD-loaded SLN, Bcl2 and HSP70 proteins were down regulated, while Bax, Bad, P53, Apaf-1, p-p53 and Noxa proteins were upregulated. This effect was also confirmed by test performed on BALB/c mice in vivo. Based on results, PD-loaded SLN may be a promising breast cancer treatment, without recognizable side effects.

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity.

Up to now, all reported Ca(2+)-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca(2+) sensitivity was lower than aequorin. Results of pK(a) calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.

Cysteine enhances activity and stability of immobilized papain.

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20 degrees C (from 60 to 80 degrees C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k(cat)/K(m)) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.

Response of salivary peroxidase to exercise intensity.

Oral peroxidase, one of the most important salivary antioxidant enzymes, is subjected to alternation due to various body conditions. The aim of this study was to assess the effect of exercise intensity on salivary peroxidase activity. Using a randomized design, ten healthy male university students (mean age, 23.22; s (x) = 2.34 years) completed treadmill runs with initial velocity 6.73 km/h at the rate of 1.58 km/h increase every 3 min until exhaustion. Unstimulated whole saliva collected over a 5-min period in pre-weighed tubes before, immediately after exercise, and 1 h after exercise was analyzed for total protein and saliva peroxidase activity. The saliva flow rate ranged from 0.08 to 1.40 ml min(-1) at rest and was not significantly affected by the exercise. Peroxidase activity in each sample was measured using 4-amino antipyrine as substrate. In the incremental exhaustion run and also at 75% VO(2max), the secretion rates of peroxidase increased. No significant changes in saliva flow rate were observed in any treadmill run. Treadmill runs at 75% VO(2max) and to exhaustion increased the activity of peroxidase immediately after exercise which decreased after 1 h. It was concluded that short-duration, high-intensity exercise increases the activity rate of peroxidase despite no change in the saliva flow rate. These effects appear to be associated with changes in sympathetic activity and not the hypothalamic pituitary adrenal axis.

Effect of Ramadan fasting on tear proteins.

Muslims abstain from eating, drinking and smoking from dawn to sunset during the holy month of Ramadan. Prolonged fasting is thought to be among risk factors for many diseases, e.g., cardiovascular, gastrointestinal and various infectious diseases. It could also play a part in several eye diseases, including dry eye syndrome, glaucoma, and cataract. Toxic and oxidative effects due to increased concentrations of some biochemicals as a result of reduction in tear volume thought to play an important role in damaging ocular tissue. Human tear is an important biological fluid similar to blood in many aspects. Tear film is composed of three basic layers i.e. lipid, aqueous and mucin. The tear film covering the ocular surface presents a mechanical and antimicrobial barrier, and endures an optical refractive surface. The aim of this study was to analyze and compare tear protein of volunteers during fasting. Using two reliable analytical methods, i.e. electrophoresis and high performance liquid chromatography (HPLC), we compared tear protein content of sixty volunteers (35 males and 25 females, 23-27 years old) during fasting in holly month of Ramadan (FAST: n = 62) and one month before Ramadan (CTRL: n = 60). The results showed that some identified tear proteins decreased during fasting. On the other hand, the activity of some enzymes such as lysozyme, lactoferrin and alpha amylase also decreased in fasting samples. Electrophoresis results showed that tear protein patterns in FAST (P < 0.05) were different from those of CTRL. There were a few more protein peaks in the FAST group (P < 0.005) than in CTRL.

A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization.

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0-10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 µmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and ß-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries.

Synergistic effect of temozolomide and thymoquinone on human glioblastoma multiforme cell line (U87MG).

AIMS: Temozolomide (TMZ) is an alkylating agent used for glioblastoma multiforme (GBM) treatment. Nevertheless, resistance to TMZ is a major obstacle to successful treatment of this cancer. The aim of the present study was to investigate the effects of TMZ and thymoquinone (TQ) on U87MG cell line. MATERIALS AND METHODS: The effect of TMZ and/or TQ on viability and invasion potential of U87MG cells was evaluated using various techniques including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase activity, cell invasion, migration, and adhesion assays. Enzyme-linked immunosorbent assay and polymerase chain reaction were used to study the expression and secretion of matrix metalloproteinases (MMPs). RESULTS: Combination of TMZ and TQ had a synergistic cytotoxic effect on U87MG cells. TMZ and/or TQ significantly reduced the potential of U87MG cells invasion. In addition, after treating with TMZ and/or TQ, there was a decrease in the levels of matrix matrix metalloproteinase 2 nad 9 (MMP 2 and 9) expression and secretion in U87MG cells. CONCLUSIONS: The combination of TMZ and TQ may emerge as a promising strategy for the successful treatment of GBM.

Molecular cloning, prokaryotic expression, purification, structural studies and functional implications of Heat Shock Protein 70 (Hsp70) from Rutilus frisii kutum.

A novel Hsp70 chaperone from Rutilus frisii kutum was identified, cloned, expressed, purified and its functional characteristics revealed. The 3D structure of Hsp70 from Rutilus kutum was constructed using the crystal structure of E. coli Hsp70 as the template, with 47% sequence identity. The in vitro ATPase activity assay after 60min, ATP hydrolysis of purified recombinant Hsp70 (8µM) was improved by binding to denatured thermally luciferase (3µM) about 2.5-fold compared with that of Hsp70 alone. Based on the results, it was found that the purified Hsp70 chaperone was able to considerably suppress heat-induced aggregation of luciferase by binding to DnaJ co-chaperone (5µM) more than 70% after 10min at 42°C. In addition, Hsp70 DnaJ complex improved the refolding of heat-shocked luciferase nearly 40% after 60min at 25°C. It was concluded that Hsp70 protein from Rutilus frisii kutum has the critical role in preventing heat-induced aggregation of luciferase and refolding of heat-denatured luciferase was strictly dependent on the activity of Hsp70, thus, this protein can potentially be used for improving the functional properties of luciferase in various applications.

Kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water and the mechanism by which glycerol obstructs the enzyme emitter site.

Renilla Luciferase is a bioluminescent enzyme which is broadly implemented as protein reporter in biology-related researches. In this study, new evidences on the kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water using MD simulation along with experimental procedures including fluorescence and CD spectroscopy were obtained. The results indicated that the Renilla luciferase activity decreased at 0.8 and 1.2 M of glycerol through the obstruction of enzyme emitter site. The present study may describe a new molecular mechanism of decreasing enzyme activity in the presents of glycerol.

Structural and functional consequences of EF-hand I recovery in mnemiopsin 2.

Mnemiopsin 2 from Mnemiopsis leidyi is a calcium-regulated photoprotein which has luminescence properties in the presence of calcium and coelenterazine. All calcium-regulated photoproteins contain EF-hand loops consisting of 12 individual residues in which the 6th position is occupied by Gly. However, the 6th residue in mneniopsin 2 is Glu rather than Gly. Here, we investigated the structural and functional consequences of substitution of Glu by Gly (E50G variant) using site-directed mutagenesis and spectroscopic procedures. It was revealed that the luminescence activity of the variant was about 17 times greater than that of wild-type (WT) photoprotein. In comparison with WT protein, our variant showed higher optimum temperature and calcium sensitivity as well as slower rate of luminescence decay. Homology modeling and sequence analysis with other known photoproteins showed that EF-hand I loop can affect the luminescence activity of E50G variant. Structural studies using circular dichroism and fluorescence spectroscopy revealed that mutation leads to the reduction in secondary structural content and local structural alterations. Finally, it can be concluded that the activity of E50G variant increases as a result of more flexibility that brought about by Gly essential for adopting the correct conformation for functional activity.

Polymorphism of MnSOD (Val16Ala) gene in pregnancies with blighted ovum: A case-control study.

BACKGROUND: Blighted ovum is one of the most common reasons for abortion during the first three months of pregnancy. Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme in the human immune system. The gene is located on 6q25 chromosome and acts on mitochondrial matrix. In the case of mutation or inactivity of this enzyme, mitochondrial and nuclear DNA will severely be destructed. The most common polymorphism of its gene is Val16Ala. OBJECTIVE: The aim was to investigate a possible mutation in pregnant women who had abortion during the first trimester of pregnancy due to blighted ovum. MATERIALS AND METHODS: In this case-control study, 34 women were entered as the case and control groups, respectively. Genome DNA was extracted from saliva samples and its genotype was determined using Tetra-primer amplification refractory mutation system polymerase chain reaction technique. RESULTS: In the case group, 16 (48%) cases had Val/Val genotype, 17 (50%) were heterozygote and had Val/Ala genotype, and 1 (2%) had Ala/Ala genotype. Among controls, 7 (22%) items had Val/Val genotype, 6 (17%) had Val/Ala genotype, and 21 (61%) had Ala/Ala genotype. The frequency of TT, CT, and CC genotypes was 48%, 50%, and 2% in case group and 22%, 17%, and 61% in control group, respectively. Statistical analysis revealed a significant relationship between Val16Ala polymorphism of MnSOD gene and blighted ovum (p= 0.0003). CONCLUSION: It has concluded that a significant relationship exists between Val16Ala polymorphism of MnSOD gene and blighted ovum.