Journal of Molecular and Cellular Researches
Year:2015 | Volume:28 | Issue:4|Papers Count:15

Recent evidence demonstrated an important role for Th-17 and FoxP3+Treg lymphocytes in immunity system. Although, previous reports have determined the immunomodulatory potential of calcitriol, but this study was mostly done before the discovery of recent lymphocytes. The present study was set out to investigate the effects of calcitriol on immunity system in NRMI-mice after challenge with sheep red blood cells (SRBC). The study population was consist of 14 male mice that randomly allocated in two equal groups and immunized with SRBC. Mice in the treatment group were intraperitoneally received 5mg/Kg calcitriol every other day from the beginning of the study and continued for 2 weeks. The results of the present study indicated a significant increase in the level of anti-SRBC antibody and simultaneously a significant decrease in the level of DTH in the treatment group compared to control group. The level of respiratory burst in phagocytic cells of splenocytes and the level of lymphocyte proliferation were significantly decreased in treatment group compared to control group. Moreover, calcitriol caused a significant reduction in the production of proinflammatory IL-17 as well as IFN-g, parallel to increasing FoxP3+Treg cells. Also the level of anti-inflammatory IL-10 was significantly increased. Therefore, the major immunomudlatoty effects of calciteriol may be due to a significant decrease in Th17 cells activity and concurrently a significant decrease in the expansion of FoxP3+Treg lymphocytes.

The photo protein aequorin, isolated from the jellyfishAequoera victoria, is a calcium sensitive bioluminescent protein formed with the apoprotein apoaequorin and the prosthetic group coelenterazine that emits light upon calcium binding. The protein is believed to be a monomer containing four helix-loop-helix motifs (EF hand) of which three domains can bind calcium. On the other hand, excess calcium in brain is associated with many neurodegenerative diseases and conditions including Alzheimer’s, Parkinson’s, and stroke. Aequorin as a natural calcium binding protein taking up excess calcium in brain cells much like a sponge. The aim of this study is production of mutant aequorin for calcium sensitivity increase. The mutated aequorin (N28D, K30T), in EF hand I, were prepared using site directed mutagenesis and subcloned in the pET21 expression vector. According to design of His-taq, purified mutants aequorin prepared using nickel affinity chromatography. Finally calcium sensitivity compared between wild-type and mutant aequorin. Calcium sensitivity of mutated aequorin indicated aeq-K30T is more sensitive than wild type and aeq-N28D.

Regarding the bacterial resistence of common antibiotecs and anti-microbial agents, a great number of studies have been conducted to discover the recent types of antimicrobial agents. With the recent developments in nanotechnology, copper has been extensively used as nanoparticles against all positive-gram and to negative-gram particles due to its low price and high anti-microbial activity. In the present study, copper oxide nanoparticles of (<20 nm) have been used to study its effect on the genome of Escherichia coli strain O157: H7 as a model for Gram-negative bacteria. To this end, the bacteria were first treated with 30 and 60 mg/mL copper oxide nanoparticles at the intervals of 2, 4, and 24 hours. and then their DNA were extracted. In order to investigate the effects of copper oxide nanoparticles on the genome, the chain reaction techniques of (RAPD-PCR) was employed. Using the software NTSYS-PC, the results obtaind from electrophoresis of PCR products on agaros gel were analyzed. The results of the studiy revealed that copper oxide nanoparticles not only affects the growth of bacteris but also affect the sequencing of genomic DNA and leads to the changes of them in different points.

In plants many genes have been identified with a significant role in the control of flowering pathway. Most of these genes belong to a large family of MADS-box transcription factors. Crocus sativus is a triploid sterile plant characterized by its long red stigmas having commercial value. Understanding flower development in this plant can be useful in increasing yield and reducing production cost. In the present study, the expression pattern of two classes of MADS-box genes (Ag1 and Sep3) involved in flower formation was analyzed in both flower parts and vegetative organs by semi quantitative RT-PCR. Based on the analysis, two C classes of MADS-box genes includingAg1a and Ag1b were expressed in three sexual organs including ovary, stigma and stamens. These two genes showed higher expression in saffron stigma and stamens compared to ovary. Expression level of Ag1a and Ag1b genes was low in petal, leaf, corn and root organs. The expression of Sep3a, Sep3b, Sep3c and Sep3d genes from class E of MADS-box family detected in petal, stamen, stigma and ovary while it was not detectable in vegetative organs including corn and root by semi quantitative RTPCR.

The goal of tissue engineering is regeneration and restoration of damaged tissues and organs. Scaffolds are the main part of tissue engineering, and gelatin/chitosan surface modification is one of the proper methods for surface modification of polymer scaffolds. Evaluation of fibroblasts behavior on gelatin/chitosan coated supramolecular PCL (SP-PCL) was the aim of this study. SP-PCL nano-fibers were prepared by electro spinning. Morphology of the nano-fibrous mats was investigated using scanning electron microscopy MTT assay was performed to examine cell activity on the nano fibers. DAPI staining was carried out to evaluate cell viability. The SEM images of the cells onto the scaffold showed good biocompatibility of the scaffold. In addition, the MTT assay results showed a proper metabolic activity of cells on the scaffolds The sPCL nano fibrous mats coated with gelatin-chitosan blend show significantly (p<0.05) increased cells proliferation compared to control (tissue culture plate, TCP), and also this significant increased proliferation has been observed until the seventh day post seeding. The experiments showed that the nano-fibrous electro spun SP-PCL coated with gelatin/chitosan blend, provides a suitable environment for fibroblasts adhesion, and proliferation, possibly because of similarity to nano-fibrous natural ECM network.

Nitrogen is one of the most important and most frequently used elements for plant growth. The need of plants to nitrogen supplied through chemical fertilizers that causes many environmental problems. Biological nitrogen fixation is one of the exceptional natural phenomena that accomplished by some of bacteria including Rhizobia. Rhizobium leguminosarumbv. viciae is the specific symbiotic nitrogen fixer of faba bean. Plant infection test is one of the inexpensive and reliable methods for detection of Rhizobium. In this research the sampling of Rhizobium root nodules of faba bean in 50% flowering stage in fields of farmers from Khouzestan, Lorestan, Golestan, Mazandaran and Gilan provinces was accomplished. A total, 168Rhizobium root nodules were collected from these areas. After isolation of bacteria from nodules, various morphological and physiological tests were performed on the isolates. Accordingly, 101 isolates were purified and detected. Plant infection test revealed that 42 isolates are able to form root nodules on fababean. The symbiotic effectiveness of selected isolates was 5-165. The main Conclusion of this research was achieving to some super strains of native Rhizobia in terms of nodulation and symbiotic effectiveness for future investigations for introducing Rhizobium inoculums for fababean.

Injectable hydrogels are among the most applicable materials in soft tissue engineering. Synthetic biology is been applied to design and produce new hydrogel biomaterials with suitable mechanical properties and in situ gel formation capability in in vivo conditions. Amphiphiles peptides are one mostly used group of these biomaterials. Biological, chemical and mechanical optimizations are necessary for a good soft tissue ECM simulation based on hydrogels. The aim of this work is to construct a novel three-dimensional nano-composite hydrogels with different mechanical and biological properties. The Spectroscopic and microscopic methods consisting of CD, FTIR, TEM, and AFM are used for optimization of the co-assembly process and to assess the probable effects of bio-epitope segments in final properties of co-assembled hydrogels. Our results show that not only the amount of each peptide but also hydrophobicity and the volume of bio-epitope segments affect the strength and geometry of hydrogen bonds, inter-febrile interactions, and final stability of hydrogels. These data suggest that it is possible to produce different hydrogels without major alterations in the main parts of peptide amphiphiles just by designing right combination of mixed peptide amphiphiles and their bio-derivatives.

In this study we use relative expression analysisvia quantitative Real-Time PCR to evaluate the effects of drought on the expression pattern of genes coding for the enzyme S-adenosyl-L-methionine: phosphoethanolamine N-methyltransferase (PEAMT) in Arabidopsis thaliana, Tissues including fast-expanding leaves, fully expanded leaves, and roots have been considered. PEAMT plays a key role in the biosynthesis of phosphocholine. According to the Arabidopsis thaliana genome database (TAIR) there is one known gene At3g18000 (AtNMT1) and two putative genes At1g48600 (AtNMT2) and At1g73600 (AtNMT3) coding for the PEAMT enzyme. The tissue mRNA induction level of AtNMT1and AtNMT3 genes varied in response to drought levels, while AtNMT2 gene expression was affected negligibly by these factors. While withholding water for 12 days reduced the expression level of AtNMT1 in fast expanding and fully expanded leaves by about 0.8 folds, the expression level of the gene increased in roots by about 4.5 folds. In contrast, after 16 days without watering, the expression level ofAtNMT1 rose about 2.5 folds and reduced to the level of control in fast expanding leaves and roots, respectively. The expression pattern of AtNMT3 suggests that AtNMT3 may be a drought responsive gene in roots. AtNMT3 expression in roots increased about 3.5 and 9 folds in response to 12 and 16 days of water deficit, respectively, despite the lack of changes and/or reduction in leaves. We conclude that the genesAtNMT1 and AtNMT3 may influence the response to drought stress in Arabidopsis.

Soil salinity is a major limiting factor of agricultural crops in the world. Increase in the level of expression of some genes that involved in salt tolerance of plants, can lead to improving performance. The salt stress induces SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana). SOS3 protein has an important role in this regulatory pathway. In this study, we have isolated the cDNA of AtSOS3 gene and cloned it into the pBIN61 vector under control of CaMV35S expression promoter. Then we have transformed Arabidopsis plants by Agrobacterium containing the recombinant plasmid, by Floral dip method. The mature seeds of transformed plants was selected on the culture media with kanamycin. The presence of the SOS3 transgene in the transgenic plant cells was verified by the PCR method. Analysis of second generation of transgenic and wild type plants for their salt tolerance in different doses (0, 50, 100, 150mM) of NaCl showed that transgenic plants are more tolerant to salinity stress.

Extensive research has been conducted on the stability of proteases in organic solvents, because they have numerous applications in organic media. Thermolysin fromBacillus thermoproteolyticusis a highly thermostable Zn-metalloprotease and is applied in industry for peptide synthesis, a reaction essentially performs in organic media. However, thermolysin has low stability at organic solvents.In contrast, its homologous enzyme, Pseudomonas aeruginosa elastase (PAE) is an organic solvent stable protease. In the present study, recombinant PAE was purified and compared with thermolysin in different organic solvents. For this purpose, activity of enzymes was measured in ethanol, methanol, isopropanol, dimethylformamide, glycerol and ethylenglycol (0-50 % (V/V)). Except isopropanol and ethanol, not only the elastase activity was not decreased, but also strongly increased in organic solvents. However, activity of thermolysin was abolished in all organic solvents. In the presence of DTT 1mM, the PAE activity was notably decreased in organic solvents. Considering that PAE contains two disulfide bonds at N- and C-terminal domains, it may suggest that disulfide bonds play a role in organic solvent stability of PAE.

From the first vitrification ofmouse embryos in 1985, considerable efforts have been made to develop and improve cryopreservation techniques, but still biochemical and fetal chromosomal abnormalities caused by vitrification can cause changes in the pattern of genes expression involved in embryonic development and function, reduced implantation and fetal death. The purpose of this study was to investigate the effects of vitrification on survival and the expression of pluripotency specific genes likeOct4 and Nanogin the vitrified-warmed mouse blastocysts. Harvested blastocysts from superovulatedfemale mice were vitrified in the presence of dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 0.5 M as cry oprotectants and vitrified in liquid nitrogen after loading on Cryotop. Thawing process was performed by immersing the blastocysts in thawing solution prepared using sucrose 1 M. Survival and hatching rates were evaluated and also theOct4 and Nanog genes expression in vitrified- thawed and fresh blastocysts (control group) were assessed by real time-PCR method. Survival and hatching rates decreased significantly in vitrified blastocysts compared with control group (94.43 ± 5.81% vs.100% for survival and 57.78 ± 11.76% vs.86.88 ± 5.80% for hatching). Also increased expression of Nanog and Oct4 genes were observed in vitrified- warmed blastocysts. Considering the results of this study, it can be said that vitrification ofmouse embryo at the blastocyst stage can change gene expression and quality of the embryo.

Therol (rooting locus) genes are located on T-DNA (Transferred DNA) of Agrobacterium rhizogenesand induce the morphological and developmental abnormalities upon the integration in to the plant genome. In this study we investigated the effect of bacterialrolC gene and its homologue in Tobacco -tobacco rolC or 'trolC' gene- on growth of Nicociana tabaccum cv. samsun plants. In the first time, the presence of rolC and trolC genes was confirmed in transgenic lines and then using RTPCR technique, we have shown that the transgenic plants expressing the bacterialrolC or tobaccotrolC genes -which demonstrate the chlorotic phenotype-, have very low or no expression of the chloroplastic gene 'psbA'. This gene encodes for one of the proteins of photosystem II. We conclude that the lack of expression of chloroplastic psbAgene and thus the defective feature of chloroplasts in rolC/trolC transgenic plants may cause the chlorotic phenotype of these plants.

Due to the widespread of environmental pollutants and heavy metals, the use of modern, fast and high performance methods for detection of the pollutants has great significance. One of the methods to assay the toxicity of environmental samples is the use of luminescent bacteria for the assay based on bioluminescence inhibition. The purpose of the current study was toxicity assay of heavy metals using luminescent bacterium isolated from the Caspian Sea. Water samples were collected from different depths of the Caspian Sea. For the isolation of luminescent bacteria, samples were inoculated into specific medium and incubated at 28 °C. Growth and luminescence of the isolated were studied using spectrophotometry and luminometry. Then the assessment of the heavy metals toxicity including Pb, Cd and Cu was performed using the luminescence property of the isolate. A luminescent bacterium was isolated from sea water and named MM1. The result of bioluminescence investigation revealed that the isolate MM1 can be able to emit a steady and high level of light. The light emission of isolate MM1 was significantly decreased in the presence of heavy metals. The results revealed that the isolate was able to detect low concentration of heavy metals. The EC50 values for Pb, Cd and Cu were assayed 8.55, 18.46 and 3.77 mgL-1, respectively. The 16S rDNA sequencing was exhibited that the MM1 strain was similar toVibrio harveyi with 99% homology. The results of this study demonstrated that the luminescence activity of bacterium isolated from the Caspian Sea was sensitive to the presence of heavy metals and was significantly reduced. The isolate can be used for toxicity assay of environmental pollutants especially for heavy metals.

Plants are constantly challenged by the invading pathogens in their natural habitat. Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. In this study, synthetic promoters were placed upstream of the chimeric chitinase defense gene to produce transformation vectors. Canola plants were transformed with three constructs, pGFC, pGMPC, pBISM2 containing synthetic promoters, SP-FF (FF elements+minimal promoter), SP-MP (only minimal promoter), and the CaMV 35S constitutive promoter, respectively. Enzyme activity assay indicated that the synthetic pathogen-inducible promoter (SP-FF) was responsive to the Methyl jasmonate (MJ) elicitor, whereas pGMPC, as a negative control did not respond. Transgenic lines were also evaluated for antifungal activity against two phytopathogenic fungi. Data show that leaf extracts from transgenic plants containing the SP-FF promoter treated with MJ, strongly inhibited fungal growth, especially that of Sclerotinia sclerotiorum, the major pathogen of canola. Results indicated that not only the SP-FF synthetic promoter is highly responsive to MJ, as an important chemical signal in necrotrophic pathogen defense, but the induced expression of the transgene under control of the synthetic promoter can increase the resistance of transgenic plants against pathogenic fungi.

Presence of missing values in microarray data decreases accuracy of drawing regulatory gene networks and may cause mistake in clustering and specialized classification of genes and further analysis. Therefore, missing values estimation is one of the most important steps in preprocessing of microarray data. Function of estimation algorithms is varied in different datasets and different missing percentage. Select a proper algorithm, in order to achieve the most accurate results in calculation of missing values, is a critical point. In this study, three microarray datasets were used. Dimensions of expression matrix was determined, and data normalization was carried out, then, different missing percentages were applied on under studied datasets.11 machine learning algorithms were used to estimate the missing values, and their accuracy were compaied based on the output. Based on the archeived results, accuracy of each algorithms depends on used datasets, missing percentage, and missing distribution. Also, the number of experimental samples in datasets can affect the accuracy of missing values estimation algorithms. The results revealed a descending trend in accuracy over increasing the percentage of missing data. However, Least Square Adaptive and Local Least Square algorithms showed more accuracy through increasing of missing percentage rather than others.