JOURNAL OF MOLECULAR AND CELLULAR RESEARCHES
Year:2015 | Volume:28 | Issue:1|Papers Count:14

NsLTPs (nonspecific lipid transfer proteins) are plant proteins subdivided into nsLTP1(9kDa) and nsLTP2 (7kDa) according to the molecular weight. These highly stable proteins can protect drugs against oxidation or degradations. Rice nsLTP2 due to the inherent ability to cross biological membranes and bind to steroid compounds (including some pharmaceutical compounds) have potential application for use in drug delivery systems. To enhance the biological capability of this protein in this system, structural alterations are indispensable. These alterations can be created by desired mutations in the encoding genes. Analysis by different software showed that the hydrophobic cavity of the active site plays a prominent role. After designing of mutagenic primers (Tyrosine 45 to Tryptophan (Y45W)) the method of SOE-PCR was used to perform site directed mutagenesis in the gene of rice nsLTP2. Wild type nsLTP2 was cloned into the PGEX6p2 vector. This wild type plasmid was used as a template for constructing the mutant fragment and designed mutant primers are used for amplification of mutant fragments. Mutant rice nsLTP2 are expressed in the pET32a vector in to the E. coli BL21 (DE3) strain. After the amplification of the mutant fragments and confirmation of mutations generated by sequencing, the mutant constructs cloned into the pET32a vector and the fusion mutant protein with a His-tag was purified by Ni-NTA affinity chromatography. The purpose was to create mutations with increased florescence signals. We hope that, in the future, these findings would be helpful in creating biosensors or as drug carrier vehicles.

Cerebral Ischemia is a neural disease which causes dying of a number of people every year. The disease usually exposures since stopping the blood pressure and also arresting of heart muscle. HMGB1 is a member of High Mobility Group superfamily proteins which all have a common DNA binding. HMGB1 is released as an extracellular signaling molecule and it binds to Toll like receptors at the cell membrane in the brain cells inflammations. Totally it leads to increase the cytokines and it also intensifies the inflammation. More inflammation can cause the brain cells apoptosis such as neurons and neuroglia cells. Rosamirinic acid, Tanshinone A and Tricin-7 Glucoside which were used for inhibition of HMGB1, they were studied using the Molegro Virtual Docker ans LiganScout softwares in this study. According to the scores, results showed that Tricin- 7 Glucoside had better physical and spatial interactions with HMGB1 and TLR2 than others and also Tanshinone A had the best interaction with TLR4.

Mixed aerobic and anoxic conditions, in addition to substrate versatility, provide a wide range of microhabitats and niches for a large number of microbial species including bacterian yeasts and filamentous fungi. Among them, study of environmental yeast species, especially those producing secretory catabolic enzymes, can be of a great importance. After enrichment of water and soil samples and their cultivation on antibiotic supplemented YPG Agar, 46 isolates were obtained. Most of the isolates were capable of producing nucleases and sterases. Using cup plate method revealed that strains SA044 and SA006 were able to produce the greatest number of secretory enzymes. Production of at least one secretory amylase, protease, nuclease and lipase was recorded in one or both of these isolates. Production of secretory keratinase, cellulose, phytase, xylanase, chitinase and pectinase was not observed. Using molecular approach and phenotypic examinations, the isolates SA044 and SA006 were identified as Pseudozyma aphidis and Pseudozyma antarctica respectively.

Aspergillus flavus is the fungi that caused massive contamination on feed. In this study antifungal activity of different extracts of Aloe Vera plant on the growth, aflatoxin production and extract effects on profile of extracellular proteins of Aspergillus flavus were study. This research conducted on Aloe vera utilizing sex solvent including acetone, ethanol, water, methanol, chloroform and ethyl ether for extraction from Aloe Vera fresh leaves. Antifungal activity of the extracts was evaluated by Agar Plate Diffusion Plate method. Each of extract in different concentrations of 0, 2, 20, 200 and 2000mL in 20mL was tested. HPLC and SDS-PAGE techniques in order of evaluation of acetone extract of Aloe Vera, and its extract effect on the production of aflatoxin B1 and extracellular protein patterns produced by A. flavus were used respectively. The maximum antifungal activity was observed in acetone extract, in concentration of 2000mL. Results obtained from HPLC analysis revealed the inhibition of aflatoxin production in 2000mL and 2mL was 40.94% and 18.14% respectively. The SDS-PAGE results showed, with decrease in fungal mycelium growth, the proteins production rate was also decreased. Generally result showed that the acetone extract of Aloe Vera can be used as a more effective antifungal agent to inhibit the growth of A. flavus compared to other solvents.

Charge, dipole moment and quadrupole moment eigenvalues as physicochemical features have been used for the RNA-binding function prediction of proteins, in this study. Simple and efficient logistic regression method was utilized for the prediction process. In the corresponding logit equation, charge feature had the highest coefficient (19.19) and impact on the prediction and dipole moment was the second significant feature. Logistic regression was trained using jackknife procedure on 2601 protein chains (160 RNA-binding proteins and 2441 non RNA-binding proteins). The value for the performance measure of area under the curve of receiver operating characteristics (ROC) was 83% for the final model and is higher than the value obtained by the neural network method for prediction. The values of accuracy, precision and F-measure were 94%, 59% and 46%, respectively, which outperformed the neural network method. In conclusion, we showed that with the help of simple, fast and accurate logistic regression method, RNA-binding proteins can be well distinguished from non RNA-binding proteins using a few number of physicochemical predictor features.

Laccases have many biotechnological applications due to their oxidation ability towards a wide range of phenolic compounds. In this study, the strain QZA2 isolated from Iran soils was used. This strain showed high similarity to Bacillus anthracis strain ATCC 14578 according to 16S rRNA gene sequence. Laccase gene was amplified by cloning primers. then PCR product cloned in the expression vector (pET21a) and transferred to BL21(DE3) strain of E. coli under the control phage T7 and sequence analysis carried out. The gene of the QZA2 has an open reading frame composed of 1656 bases, which encodes 551 amino acid residues and contain four histidine rich copper-binding domains. The laccase gene from QZA2 showed 16% similarity with CotA from B. subtilis and 57% similarity with multicopper oxidase B. halodurans. The expression was performed under microaerobic condition in order to obtain high amounts of soluble protein. Biochemical properties were investigated using common laccase substrates ABTS.

The enzyme acetylcholinesteraseinsensitivity plays a major role in organophosphate insecticides resistance in T.Urticae (Acari: Tetranychidae). In this study, toxicological and biochemical resistant mechanisms to chlorpyrifos in three populations of spider mites were surveyed. Mites were first collected in Isfahan (ISR), Yazd (Yz) and Guilan (GUS2) provinces and identified based on mitochondrial cytochrome oxidase sequences. The bioassay results showed that resistance ratios of ISR and Yz populations to chlorpyrifoswere 176.9- and 9.78-fold compared to the GUS2 population, respectively. Determination of acetylcholinesterase activity with different substrates showed that resistant and susceptible strains have the highest and the lowest specific activity, respectively. In addition, AChE zymogram analyses were investigated in all populations. Lower acetylcholinesterase electrophoretic mobility in ISR population and high acetylcholinesterase band intensity of ISR population in presence of chlorpyrifosoxon showed that resistance population has altered AchE. For determining the role of acetylcholinesterase insensivity in resistant mechanism, inhibitory effectof 11 inhibitors including OPs and carbamates on this enzyme were investigated in this study.IC50 of all inhibitors on acetylcholinesterase from ISR and Yz populations were higher than GUS2 population respectively. The results suggest that some modification occurred at the active site of the enzyme that reduces the AChE affinity to substrates and subsequently led to differences in electrophoretic mobility and insensivity of enzyme to OPs and carbamate inhibitors as competitive inhibitors.

In this study, the antibacterial properties of probiotic Lactobacillus strains isolated from human mouth on Aggregatibacter actinomycetemcomitans ‘prevalent etiologic agent of periodontal diseases’ was investigated. Among 40 Lactobacillus strains isolated from healthy and diseased human mouths, the most efficient antibacterial-producing strain was selected for further studies. The morphological and biochemical characterization of selected isolate matched the literature description about genus Lactobacillus. Partial sequencing of 16S rRNA gene and its alignment with other Lactobacillus strains revealed that the isolate was closely related to the Lactobacillus salivarius. We thus tentatively labeled the isolate as Lactobacillus salivarius NK02. The study of probiotic properties of NK02 strain demonstrated that the isolate had significant resistance to lysozyme and adequate ability to grow in the presence of bile salts. Viability of the NK02 strain in simulated gastric juice was remarkable and the survival rate of isolate was 7 logs CFU ml-1 after 90 minutes. The NK02 strain was sensitive to most antibiotics and had anti-bacterial activity against certain pathogenic bacteria used in this study. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of HK01 strain against Aggregatibacter actinomycetemcomitans was determined as MIC: 512 AUml-1 and MBC: 1250 AUml-1 that was higher than expected compared with other Lactobacilli.

Arthrospira platensis microalgae have many applications for human and animal as food and drug because it has more essential nutrition than other microalgae. It is a microscopic filamentous alga that is rich in proteins, vitamins, essential amino acids, minerals and essential fatty acids like y-linolenic acid (GLA). It is produced commercially and sold as a food supplement in health food stores around the world. In this research mass culture of spirulina has been done in 30oC by Jourdan medium in green house, when spirulina arrived to maximum growth; it has been harvested by Micronics net and dried in room temperature. Dried spirulina have been kept in refrigerated until microbial analysis. In next phase, fresh spirulina and spirulina powder have been cultured on blood agar media according to the standard method. Then total coliform has been counted in both samples. According to standard documents these spirulina is not good for human but we could use them for poultry and fish food.

Iran is known as one of the early centers of Safflower. Therefore, the diagnosis diversity safflower genotypes for maintain genetic resources and scientific and practical applications of these materials may be useful in breeding programs for breeders. This study investigated the genetic diversity of 24 genotypes germplasm international, Iranian landraces and wild safflower using morphological traits and RAPD markers was conducted. The results showed that a high diversity of morphological traits in safflower genotypes, this diversity for safflower breeding programs that can appropriate manner be used to increase the yield. The study of molecular markers by 11, multiply in the 142 bands, 84 bands were polymorphic (57.7%). The results of cluster analysis showed that molecular markers are unable to detect the genotypes from one another. Iranian accessions were present in most of the groups that the reason for the high diversity within the population of Iran and Iran candidates one of the main origins and very likely is for safflower germplasm. The results of this study showed that the combined use of morphological and molecular markers for safflower breeding program is especially helpful for selecting parents for the hybridization and the performance RAPD molecular markers is to study genetic diversity and germplasm management safflower. Furthermore the Iranain local populations of safflower and wild safflower are a rich source of genetic for use in breeding programs safflower.

Trichoderma harzianum crude extract was prepared and its controlling effects on a wide range of Gram positive and negative bacteria and a few fungal species were evaluated via disk assay techniques. The extract was able to control pathogenic bacteria including Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Salmonella typhi, Staphylococcus aureus, two Xanthomonas citri pathovars (NIGEB-88 and NIGEB- 9322), and also two fungal pathogens: Macrophomina phaseolina and Rhizoctonia solani. However, it failed to control a pectobacter, Erwinia amylovora. The biocontrol efficacy of crude extract was lost once treated with trypsin and reduced in the presence of SDS, indicating the protein nature of the effective materials in the fungal extract. The antimicrobial protein was resisted 100oC for 30 min and was active in a wide range of pH (4-9). Our data is indicative of an efficient isolation of novel fungal thermostable antimicrobial proteins with the potential to be used as a biocontrol agent in the future.

Urmia Lake is one of the hyper saline Lake on world which has been faced with drought crisis recently. Increasing salinity up to saturation, the sharp reduction in the density of unicellular and multi-cellular algae as well as Artemia are effects of the crisis. Despite this critical situation on the lake, in summer of 2012 a very strong algal bloom was seen at northwest Urmia Lake (Bari station). Analysis of the chemical compositions of the water and study of the algae species were provided on the obtained samples from that area. Molecular analyses were provided by study of 18srDNA gene and sequencing of ITS region. The results of this study revealed the dominant species of the algae as Dunaliella tertiolecta which was found in concentration of 1.2´106 cells/ml in salinity above of optimum. Also high levels of certain minerals such as phosphate, ammonia, nitrate, as well as salinity stress in the area was proposed as possible causes of algal bloom.

Cell-therapy provides a promising alternative for the treatment of type 1 diabetes. One of the most proper candidates is multipotent skin-derived precursors cells (SKPs), which can be differentiated into insulin producing cells (IPCs). In this study, human skin-derived precursors (hSKPs) were isolated, expanded and differentiated into IPCs in vitro, through exposure to rat pancreatic extract (2 days after a 60% pancreatectomy). In order to differentiation, SKPs were Cultured in DMEM, containing FBS, pancreatic extract and glucose for 14 days. In order to verify insulin production in the cells, dithizone-staining, which is a method for insulin identification, was employed. In addition, insulin expression was examined immunocytochemically, and insulin secretion was examined using enzyme-linked immunosorbent assay. Cellular clusters appeared after approximately 14 days. The clusters were found to be immunoreactive to insulin. Cellular clusters were also able to secrete detectable amounts of insulin in glucose concentration dependent manner. The results of the current study showed that pancreatic extract treatment can differentiate human SKPs into functional IPCs. This may offer a safer cell source for future stem cell-based therapies.

Haraz is one of the three full of water rivers in north of Iran (Mazandaran province) that emanates of northern slopes of the Alborz and collects pollutants in their path with passing through agricultural areas, cities and villages and finally discharges to the sea. The aim of this study was to evaluate the microbial quality of surface waters of Haraz river and microbial indicators such as total and fecal coliforms. In this study, 84 samples were sampled of surface waters of Haraz River from seven stations (Sorkhrood, Karesang, Jalav Bridge, Norroud, Tehran 115, Lasem, Lar (polour)) during one year (a total of 12 samples) and were examined the coliforms and fecal coliforms. The results showed that maximum and minimum of total coliforms and fecal coliforms in surface water was observed in Sorkhrood (9.4 CFU/100ml and 3.2 CFU / ml) and Lasem (4.2CFU/100ml and (1.1CFU/100ml) respectively. The results showed that the populations of indicator bacteria (Coliforms) have been different depending on seasonal changes, time and sampling of location. The final results of this study showed that the river quality is low and not suitable for human consumption.