Journal of Molecular and Cellular Researches
Year:2013 | Volume:26 | Issue:2|Papers Count:8

Glycine-Rich RNA Binding Protein2 (GR-RBP2) is one of the eight GR-RBP family members in Arabidopsis thaliana which is located in mitochondria after translation. Here we employed a proteomic approach to compare transgenic Arabidopsis plants over expressing GR-RBP2 with Wild Type. In this study, GR-RBP2 gene was isolated from Arabidopsis and cloned into pBIN19 vector. Recombinant vector was transferred to Arabidopsis (Col-0) via Agrobacterium tumefaciensby Floral Dipping method. Transformation with full length cDNA of GR-RBP2 yielded 21 lines. Over expressing plants are indistinguishable from WT seedlings. The whole lines are unchanged with regard to flowering time and are fully fertile. Gene expression levels were determined in different over expressing lines compare with WT seedlings. The mRNA level in over expressing lines was 2 times higher than WT. Proteomic analysis of over expressing lines were carried out on two different gel systems: 2D IEF/ SDA PAGE and 2D Blue-native/SDS PAGE. All gels were silver stained. 2D gel electrophoresis revealed that GRRBP2 was induced in over expressing lines. Results showed that respiratory chain looks very much in the same investigated lines and WT. Complex V was partially degraded (F1 complex), but this also is the same in all samples. Approximately 110 spots were identified by mass spectrometry. Six protein spots were increased in over expressing lines compared to WT. A look at some well known proteins showed that there is no major change indicating induction or repression of metabolic pathways.

The Crocus genus, which comprehends approximately 80 species, is known mainly for the cultivated species Crocus sativus. Genetic variability among 16 genotype of Crocus genus originating from Iran was examined for the first time with Inter Simple Sequence Repeat (ISSR). Fourteen primers generated a total of 208 discernible and reproducible bands across the analyzed populations, that all of these showed polymorphism. The fragment scored for presence/absence and used to generate Dice, Jaccard and Simple Maching similarity coefficients and to construct a dendrogram by means of UPGMA and Complete Linkage in NTSYS-pc 2.02 computer program. Cluster analysis revealed primarily five major groups. Furthermore, dimensional graph derived from Principal Coordinate Analysis (PCoA) of ISSR data also revealed a pattern in which the genotypes were assigned into five separate groups. Estimation of Resolving Power (RP) values exhibited a collective rate of 134.08 and varied from 4.87 for UBC 872 to 15.5 for UBC 851 with a mean of 9.6. The results showed the high effectiveness of ISSR markers in grouping of Crocus species under study, and analysis of their genetic relationships.

Luciferase enzymes efficiently convert luciferin to oxyluciferin in the presence of ATP, Mg2+ and molecular oxygen. Luciferase reaction produces a spectrum with different color under various conditions. One of the most important factors is residues of structure. Lampyris turkestanicus, Iranian firefly, luciferase emits green light. In order to study, 3D-structure determination and red-shift mechanism analysis in comparison to other determined structure luciferase, Mutated (E354R/R356/H432Y) (emits red light) luciferase from Lampyris turkestanicushas been crystallized. The crystals with 2.2Å resolution of mutated firefly luciferase were obtained by the sitting drop method. The diffraction pattern from E354R/356R/H432Y luciferase belongs tetragonal system with space group P41 and unit cell dimensions a=b=85.01 and c=97.09.

Pectic substances are complex polysaccharides, rich in galactoside residues, capable of combining with the carbohydrate-binding domain of galectin-3. It has been shown that these biomaterials can induce apoptosis in tumurigenic cells, possibly via interaction with galectin-3. In the present study the apoptotic effect of pectic acid (AP) and citrus pectin (CP) on the human prostate cancer cells DU145, which express galectin-3, is investigated. PA and CP were modified by alteration in the temperature and pH, and the apoptotic effect of such modified CP (MCP) and AP (MAP) was studied. We also used commercially available modified citrus pectin, Pectasol, as positive control. DU145 cells were treated by different concentration of AP, MAP, CP, MCP, and Pectasol in periods of 24 and 48 hours. The percentage of apoptotic cells, after these treatments were investigated using Acridine orange/ Ethidium bromide staining and Cell cycle analysis. These investigations showed that AP, MAP, MCP can induce high percentage of apoptosis compared to control group (p<0.001). In addition, fluorescent staining showed that percentage of apoptotic cells were higher than necrotic cells. We demonstrated that, AP without any modification induced apoptosis, yet CP should be modified to have such value. It may be a dominant point for AP as an edible pectin, while citrus pectinase is in the non-edible peal and should be modified to have such property.

Unicellular ciliates including Paramecium caudatum can be cultivated in yeast medium. In this research the effect of temperature and medium enrichment on growth and reproduction of the ciliate population was evaluated. The growth of the P. caudatum population in the ordinary yeast medium under a range of temperature from 20 to 40°C and enrichment of the medium using both the ions (Ca2+and Mg2+) and the L-Arginine were compared. Based on the results the population of the ciliate 48 h after the cultivation in the ordinary yeast medium increased at a significant level (p<0.001) under 30oC compared with those of other temperatures. Moreover, the enrichment of the medium with ingredients (Ca2+and Mg2+) especially after adding of L-Arginine under optimum temperature caused more increase in the population of the ciliate to that observed in the ordinary medium (p<0.001). The growth curve of P. caudatum in enriched medium under optimum temperature showed 4 phases. A comparison between growth curves of this with that of ordinary medium indicated much upward growth in the enriched one. According to this work an enriched yeast medium under the temperature of 30oC can be introduced as a suitable artificial medium for cultivation and proliferation of P. caudatum.

Rosa damascena Mill. is one of the valuable species with a long history in Iran and some other countries. In order to investigate cytogenetic variation in Rosa damascena, meristemic zone of 14 accessions were used. The research was conducted using a completely randomized design. Image analysis system and Micro-measure software were used to karyotype providing and cytogenetic parameters measurement. Data were collected and analyzed using a completely randomized design with three replications. Results showed significant differences (P<0.01) among accessions for some parameters, such as short arm (SA), long arm (LA), arm ratio (AR), Intra asymmetry chromosomal index (A1) and total form percentage (TF%). Using principal components analysis, the first three independent components accounted over 98% of variation. The first principal component indicated that short arm (SA), arm ratio (AR), intra symmetry chromosomal index (A1) and total form percentage (TF%) are appropriate parameters to classify accessions with 51% of total variation. Long arm percentage and Short arm percentage were important traits in second component at 24% level. Long arm and Total length (TL) were important traits in third component at 22% level. Cluster analysis based on cytogenetic parameters, grouped the accessions into 4 categories. The most far apart accessions were Isfahan 9 and Ilam1 and the most near ones, Gilan 1 and Isfahan 7. It is necessary to notify distribution of accessions based on the first three component scores were in agreement with cluster analysis.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea in children under 5 years and it is also a cause of Travelers diarrhea worldwide. To prevent ETEC-caused diarrhea and decrease its prevalence, the WHO has promoted the vaccine production against this pathovar. CFA/I fimbriae is an important and frequent virulence factor of this bacteria, playing a critical role in pathogenesis. Therefore, tip protein of this fimbriae (CFaE) could describe as a vaccine candidate. This study was aimed at investigating the expression of the gene rCFaE from native sequence after cloning into expression vector. rCFaE was amplified by PCR using a newly designed set of primers. PCR product was then cloned into early cloning vector pTZ57R/T and then sub cloned into the expression vector pET28a(+). Protein expression in 2 strains of E.coli BL21-(DE3) pLysS and Rosetta was determined by using IPTG under different conditions. cloning and sub cloning process were performed successfully. Test samples in the comparatives with control samples did not show detectable protein on the SDS-PAGE. The same results were obtained after changing different parameters like time and temperature of induction and variety of IPTG concentration. It was not possible to obtain high level expression of the target recombinant protein production in E.coli with pattern codon usages, probably due to the high A+T content and also abundance rare codons in the native sequence of cfaE gene. Therefore we suggest synthesizing this gene after codon optimization and checking for expression that again.

By attention to this fact that bacterial lipase has important role in different industries, in present investigation potential of Pseudomonas aeruginosa from burn infection for lipase secretion was studied. Optimization procedure using response surface methodology (RSM) based on Box-Behnken Design (BBD) with four factors (Ca2+ion, olive oil and tween-80 concentration and incubation time) was used in order to investigate the effect of these parameters on the production of lipase from newly isolated bacterium; Pseudomonas aeruginosa HR59. According to the results of RSM, concentrations of Ca+2, tween-80 and incubation time were very important role on enzyme production. As a result of this optimization, maximum lipase activity was achievable at Ca+2 (3 mM), olive oil (1.5 % V/V), tween-80 (0.4 %V/V) and incubation time (72 h).