مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال بیست و هشتم شماره 2 (تابستان 1394)

Asparaginase is a hydrolytic enzyme that catalyses asparagine to aspartic acid and ammonia. The enzyme is used for treatment of acute lymphoblastic leukemia (ALL), lymphoma and melanolymphoma. S. marcescens is a Gram-negative bacterium with red pigment that can produce asparaginase in culture medium culture in the presence of asparagine. In this study asparaginase was purified and kinetic parameters of enzyme were determined. A modified culture medium was used for enzyme production containing malt extract, soy peptone and asparagine. Enzyme activity was detected after 8 hours and maximum enzyme activity was reached after 40-44 hours of incubation. Purification of the enzyme was carried out using ammonium sulfate precipitation. The concentrated enzyme preparation was loaded onto DEAE cellulose chromatography column. The SDS-PAGE was determined a single band with about 54 kDa protein. The optimum pH and temperature of enzyme activity were determined to be about 7 and 40˚C respectively, and the kinetic parameters such as Km and Vmax were measured for the first time in this study.

The bacterial expression system has the ability to produce high amounts of recombinant proteins, but the produced proteins might not have suitable structure folding and post-translational modifications. In the other hand, the mammalian cell expression system also provides the suitable form for protein production; however the higher costs and generally low production yielding for this expression system made the researchers to have tendency to study on the yeast expression system. Yeasts are particularly suited to expression of foreign proteins for numerous features, including easy genetic manipulation, high levels of intracellularly or extracellularly protein expression, and the ability to carry out higher eukaryotic protein modifications. The methylotrophic yeast Pichia pastoris has been developed for the production of various recombinant proteins and growth into high cell densities in an inexpensive medium. Secretion of low amounts of yeast endogenous proteins in cell culture media makes the simple purification of expressed recombinant proteins. However, the Pichia pastoris Glyco-switch strains with the ability to produce eukaryotic glycoproteins with the same post-translational modifications fixed the only deficiency in yeast expression system. Accordingly, Pichia pastoris is an appropriate secretory system for obtaining larger quantities of correct products, in compare to other host cells.

Currently, in silico methods considered as one of the least expensive and fastest ways for drug design and discovery in the field of therapy. In the present study, we were interested to introduce compounds, which have an important role in inhibiting tubulins polymerization as the main factor of cell division in cancerous cells using computational drug design, especially molecular docking method. For this purpose, chromene compounds, which their anticancer properties had been tested by several researchers, were gathered and based on their binding site of tubulin, new analogs were designed. Then, by using AutoDock Vina software, stronger compounds which had the lowest affinity energy, were identified and their possible interaction with the colchinine binding site analyzed by LigPlot and UCSF Chimera. Given that newly designed compounds docked to the protein structure with lower affinity energy than colchicine as the control sample, they could be the subject of further assessment as the potential pharmaceutical compounds.

Genetic diversity of Betula pendula, which is a medicinal value and endangered species, in small populations remaining in profile of northern Iran including: Marmishoo, Siahmarzkoh, Sangdeh and Shahrestanak, studied by polymorphisms of the three (3800, 1800, 2700 bp) regions of chloroplast DNA and PCR-RFLP technique. These regions named to CD, DT, K1K2, respectively. The percentage of polymorphic loci, expected heterozygosity (He) and Shannon’s information index (I) in four populations, calculated to 24.51%, 0.096 and 0.14, respectively. Analysis of molecular variance (AMOVA), revealed that 66% of total variation was found within populations, while only 34% among populations. Genetic differentiation index (Gst= 0.206) revealed a high differentiation of birch populations to each other. Pair-wise comparison of genetic differentiation showed that, Marmishoo's population by highest level of differentiation from other populations. Assessment of gene flow among populations indicate the occurrence of genetic drift between them (Nm= 0.96). In addition, Mantel test revealed a significant correlation (r=0.77) between genetic and geographical distances. Our results confirmed that the occurrence of genetic drift in birch populations, and the extinction risk of this species. Which emphasize to deployment fast and convenient strategy to it's protect.

Bacterial lipases are members of α/β hydrolase family that hydrolyzed triacylglycerol at the water- lipid interface. Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that shows optimal activity at 60–75 oC and pH 8–10. BTL2 is an important research target because of its potential industrial applications. At the present study chimeric Bacillus thermocatenulatus lipase contain the consensus sequence of Candida rugosa lipase (207Gly-Glu-Ser-Ala-Gly211) at the nucleophilic elbow region was cloned and expressed in E. coli as secretion protein. Finally, Catalytic activity of chimeric lipases was evaluated at presence of various triglycerides as substrates and the effects of different parameters such as temperature, pH, detergents, organic solvents and metal ions were evaluated on chimeric enzyme activity using a pH-stat assay. The results showed that the chimeric enzyme is most active to C4 substrate, (pH 9.0) and 60 oC. As well as enzyme activity has increased in the presence of organic solvents, N-Hexane, N-heptane, methanol, chloroform and detergents such as Triton X -100, Tween 20, Tween 40. Also, metal ions, respectively decreased general effect on the enzyme activity in chimeric lipase.

Nonspecific lipid transfer protein 2 (nsLTP2) is among prolamin super family, capable of transferring various lipophilic molecules such as sterols. Despite advantages of the protein in drug delivery systems, there are some reports on its allergenic properties. Comparative sequence analysis displayed at least 51% homology in different species of rice-nsLTP2. The aim of the current study was to evaluate gene sequence and protein structure analysis of Iranian rice-nsLTP2, by means of bioinformatics tools, to improve its potential capability for drug delivery systems. Gene sequence homology assessment revealed resemblance mostly with Japonica species (Accession NO. NP-0011048723.1). The presence of various specific sites and motifs in the structure of nsLTP2 were determined by sequence analysis. Preliminary studies demonstrated four antigenic determinants in the primary sequence of the protein, which three of them retain after post translational modification. Moreover, sequence assessment revealed that those residues contributing in the protein cavity are conserved totally among Japonica and Iranian species. Modeling approaches were used to determine the influence of deletion of allergenic sites on protein affinity toward hydrophobic ligands. The obtained results revealed the efficacy of nsLTP2, as a drug carrier protein, could be improve by appropriate swap at specific residues and elision of allergenic sites.

Today, the rats are considering as an important laboratory model and from another aspect, as very important potentially dangerous pest. Rats reseach, especially to identify them, their genetics of populations and phylogeography, using morphological and especially molecular techniques is a necessity. This study performed using D-Loop region of mtDNA to identify them and study genetic of Tehran metropolis rats and the results showed presence of only the black and brown rat species in Tehran. Among the 229 samples, only one sample was the black rat and remians diagnosed as the brown rats belonging to two different subspecies probably. Demographic and population genetics analyses showed that these animals have a very low genetic diversity and the two different groups have entered Iran in different times: The estimated time for the first or newer group and for older group was about 700 and 5600 years, respectively. The data also revealed that Tehran's rat populations have had an expansion period in past but one could see the well-done controls in the past, too. The districts such as 18, 19, 13, 12, 9, 4 and 1 have much more groups of rats and could be the niche of colonies.

Application of biorefinery approach in which in addition to bioethanol, simultaneously one or more other biological products are produced, has attracted attention as a new and hopeful strategy to economize ethanol production from lignocellulosic biomass. So, objective of the present study was to optimize co-production of ethanol and xylitol in a co-culture system of two Saccharomyces cerevisiae and Candida tropicalis strains in a batch fermentor. To determine the best nitrogen source, two strains were co-cultured on the medium containing one of 8 different organic and mineral nitrogen sources (6 g/l). The results showed that different nitrogen sources showed significantly different biorefinery efficiency, and the maximum ethanol (24.41 g/l), xylitol (22.7 g/l) and biomass (13.24 g/l) production was observed when yeast extract was used. At the next step, effect of different dissolved oxygen concentrations, 5, 10, 20 and 30%, was evaluated on the efficiency of ethanol and xylitol production at batch fermentor level. The maximum ethanol production (38 g/l) and yield efficiency (Yp/s= 0.47 gram ethanol per gram used glucose) was achieved when dissolved oxygen concentration was 5%, whereas the maximum xylitol production (21.6 g/l) and yield efficiency (Yp/s= 0.54 gram produced xylitol per gram used xylose) was achieved when dissolved oxygen concentration was 10%. Also, the maximum biomass (25.7 g/l) was achieved at 30% dissolved oxygen concentration.

Extraction temperature and solvent type were altered in order to investigate their individual effects on the biochemical compounds and antioxidant capacity of Artemisia absinthium. Therefore, alternations on biochemical composition and antioxidant potential of plant extracts were studied in response to temperatures (30-60 °C), solvent types (methanol, ethanol and acetonitrile) and solvent concentration (25-100%). Total phenolic content (612-1033 mg GAE/100 g DW) and total flavonoid content (239-392 mg CE/100 g DW) were measured using the appropriate chemical procedures. The antioxidant tests including DPPH (51-79%), FRAP (151-287 µM TE /100g DW) and ABTS (17- 39 mM TE/g FM) were performed in triplicate experiments. The results indicated that methanol was the solvent of choice for subsequent extraction procedures. On the other hand, according to HPLC results the highest anabsinthin content was obtained when 75% methanol (16.58 µg/g DW) was used as extraction solvent, while it was lowest by 25% methanol (9.45 µg/g DW).

One way to absorb heavy metals in addition to chemical methods is the use of biological elements. One of the metal adsorption proteins is metallothionein.Goal: The purpose of this study is gene cloning and expression of Synechococcus PCC7942 metallothionein smtA and protein production and analysis of metal uptake by the protein. The gene smtA was cloned into the T / A cloning vector and then were transferred into the E.coli (DH5α). Colony-PCR evaluates the accuracy of the transformation and sequence detection was carried out. The gene was subcoloned into the expression vector pET15b and transferred into the E.coli (BL21). Gene expression was induced by IPTG and proteins were analyzed by Tris-Trisin SDS-PAGE and Western blotting. Metal uptake of protein was measured by atomic absorption. Sequencing, confirmed the accuracy of the transformation. SDS-PAGE and Western blot analysis confirmed protein expression. Metal absorption showed that one milligram of protein adsorbs 28 nmol of cadmium ion.

Pyrazinamide is one of the most important drugs in the treatment of latent tuberculosis infection. PncA gene encodes Pyrazinamidase (PZase) enzyme of Mycobacterium tuberculosis that is responsible for conversion of Pyrazinamide (PZA) to Pyrazinoic acid (active form). In spite of the role of this drug in shortening of the treatment period from 9 months to 6, the emergence of strains resistant to PZA represents an important public health problem. Pyrazinamidase mutations are associated to PZA – resistant phenotype. However, the relationship between mutations and structural changes that inactive the enzyme, has not been known very well. In this study, the PZase gene from the H37Rv strain (wild type) and two resistant strain of Mycobacterium tuberculosises to PZA were cloned, expressed and their activity were investigated by the method based on Wayne test. The structures of wild type enzyme and mutants (D63G/W119C and T160P) were modeled using homology modeling and single amino acid replacement, then structural parameters were calculated. Enzyme activity determination results revealed that the mutants lose their activity, completely. Computational results also confirmed that mutations affect the secondary structure of the enzyme and induce structural changes. These changes can alter the structure of the enzyme active site in D63G/W119C or shorten opening of the substrate binding site in T160P.

Chitinases have the ability of chitin digestion that constitutes a main compound of the fungal cell wall, insect exoskeletons, and crustacean shells. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against phytopathogenic fungi. The Chitin-Binding Domain (ChBD) of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, T. atroviride Chit42 using SOEing PCR with overlapping primers. The chimeric fragment was cloned into prokaryotic expression vector (pET26b+) and transformed to E. coli BL21-DE3.Culture conditions for chimeric enzyme production by E. coli were optimized by Taguchi orthogonal array experimental design methodology. This approach facilitates the study of interaction of a large number of variables spanned by factors and their settings with a small number of experiments leading to considerable saving in time and cost for the process optimization. The objective of the current research was to determine the significant parameters on the production of chimeric chitinase enzyme in the culture. The process variables were IPTG concentration, incubation time, and temperature. The total protein extraction of all experiments were carried out and analyzed by SDS-PAGE, Total lab and Qualitek-4 software. The optimal levels of the different factors for chimeric chitinase production were 1mM IPTG, and 16 hours of incubation time at 28 °C. IPTG concentration was the most important factor in the enzyme production.

Established by Cyrus the Great in the 6th century BC, Pasargadae is the first dynastic capital of the Achaemenid Empire. One of its most important stone monuments is the tomb of Cyrus the Great (559-530 BC) which was built with large pieces of limestone. Pasargadae is generally identified as the second largest archaeological site after the imperial palace complex of Persepolis. Today, this monument is entitled by UNESCO as a World Heritage Sites.This study aimed to survey the role of fungi in biodeterioration of this tomb stone monument for the first time. In general, 33 fungal isolates were attained from stone surfaces of the Cyrus tomb. All the isolates were subcultured onto Potato Dextrose Agar and Sabouraud Dextrose Agar. For microscopic observations, slide culture technique was carried out. The isolates were identified based on macro- and micromorphological characteristics. Overall, dematiaceous hyphomycetes and yeasts were isolated. Among the isolated fungal genera, Alternaria sp., Cladosporium sp., Ulocladium sp., Fusarium sp., Humicula sp., Arthirinium sp. and yeasts were dominant. To observe the biodeterioration process caused by fungi, Scanning Electron Microscopy was used for stone samples. The results confirm biopitting, sugaring and etching damages caused by growth of fungal hyphae on and inside the stone surfaces. The identity of the isolates will be examined by molecular sequence level data. Isolation and characterization of biodeteriorants are the first step for conservation of any stone surface of monuments.

Gastric cancer is one of the important morbidity factors in the world. There are many unique plant species that should be explored for anti-cancer components. Goal: In this project, anti-cancer effects of aqueous and ethanolic extract of Persia rose (Rosa damascena mill L.) against human gastric cancer cell line (AGS) was studied. Experimental procedure: Eight different concentrations of extracts as well as 5-fluorouracil was applied on the cells. The toxicity of the extracts, their inhibitory effects on proliferation and apoptotic effects were studied by MTT, BrdU and TUNEL assays respectively. The results showed that all concentrations of aqueous and ethanolic extracts reduced viability of the AGS cells significantly. The IC50 of these extracts on AGS cells was determined 2.517 and 3.887 µg/ml respectively.BrdU assay also showed the inhibitory effect of extracts on proliferation of AGS cells (in comparison to fibroblast cells). The proliferation of AGS cells was decreased as the concentration of both of the extracts was increased. Also, the toxicity and anti-proliferative effect of ethanolic extract was more than aqueous extract. The rate of apoptosis was determined 90% for both aqueous and ethanolic extract. Aqueous and ethanolic extract of Rosa damascena mill L. reduced viability and proliferation and induce apoptosis in human gastric cancer cells through different pathways.

In this study conditions for transformation of Datura metel was optimized. T-DNA from pZM1047 containing GUS and NPTII genes was transferred to Datura plant. Bacterial suspension was used for co culture with leaf segments., the they were transferred to MS medium supplemented with 1 mg/L BAP as regenerated medium. After 15-20 days calli with regeneration marks was appeared. The whole regenerated transgenic plants on kanamycin were checked using PCR. Transformation rate was about 13% which is 2% higher than previous study reported by others. In this study conditions for transformation of Datura metel was optimized. T-DNA from pZM1047 containing GUS and NPTII genes was transferred to Datura plant. Bacterial suspension was used for co culture with leaf segments., the they were transferred to MS medium supplemented with 1 mg/L BAP as regenerated medium. After 15-20 days calli with regeneration marks was appeared. The whole regenerated transgenic plants on kanamycin were checked using PCR. Transformation rate was about 13% which is 2% higher than previous study reported by others.