Modares Journal of Biotechnology

Abstract

Abstract Bacteriorhodopsin (bR) is a membrane protein that acts as a light-driven proton pump in Halobacterium salinarum. This protein contains seven transmembrane α-helical subunits, helices A–G, one beta-sheet and a retinal chromophore. Studies show that bR have the property of absorbing the microwave. Among several methods molecular dynamics simulation (MD) is the most systemic approach. With this method we can study structural changes and dynamic of macromolecules. In this project, we use modeling and molecular dynamic simulation. To obtain more accurate structures after the equilibration a 15 ns MD simulation was done. After that, in order to find the effective sites of microwave absorption on bR a production run was performed with applying electric field in the time intervals of 786 ps that is equal to one sinusoidal frequency at microwave spectrum. At last, conformational changes under effect of sinusoidal wave has been assigned the effective sites of microwave absorption in the protein. Our study shows that microwave in the frequency of 8 GHZ and the time interval that mentioned above, cannot make significant changes on the protein. In the other hand, we have seen some reversible changes in Beta-sheet and D, C, B helices.

Abstract Selco enriching emulsion includes compounds from certain oils with marine and herbal origins. Selco oil produces micro-globules with less than 0.1 micron diameter. In order to generate such stability in original selco chemical emulsifiers have been used up to 3%. In this study, persian gum )Amygdalus scopariaspeech( and salep (Orchis mascula (were used as herbal emulsifiers in synthesis of Artemia enriching for a content of 11% to establish emulsification. The soluble part of persian gum and salep was separated with 30% and 22% content, respectively. Through chemical analysis, the chemical composition of the imported Selco oil was identified. Then the similar ingredients of the commercial brand were combined to produce our synthesized production. In physical computing, the average diameter and distribution of oil phase particles side was obtained as 0.1 micron and the relevant surface tension as 15±5 DIN/cm. Then the synthesyzed enriching oil (treatment 1) with the imported one (treatment 2) were tested for Artemia urmiana enriching with standard conditions. Enriching conducted in 3 repetitions with 0.4 gr/lit of the enriching oils/1 lit of water. Nauplii were introduced as 200000 nauplii/lit. The average of nauplii enriching percentage in treatments 1 and 2 was achieved as 27±2.47 and 23±2.52 percent, respectively. The bioassay results on 500 new feed larvae of trout fish has been shown that treatments 1 and 2 were significantly differed in survival. Therefore, the plant emulsifiers in this study, showed good performances as the chemical and physical properties, in stabilizing the oil emulsion in the aqueous phase.

Abstract Background: Campylobacter rectus is one of the important periodontopathogenic bacteria in many regions of the world. According to lack of published surveys on the bacterium in Iran, the aim of this research was to detect of Campylobacter rectus in the patients with periodontitis referred to dental clinic of Tehran University using molecular method. Materials and Methods: Sampling from 40 patients was performed by clinic specialists at deepest site of periodontal pockets using paper points and corresponding data were registered. DNA of the specimens were extracted using DNP TM kit (Cinnagen) and PCR was carried out using C. rectus specific primers and ATCC 32238 reference strain as positive control. Statistically significant differences in frequency of bacterial contamination and mean depth of gingival sulcus among different age and sex groups were evaluated using SPSS 22 (Chi square and t-tests). Results. Molecular method results showed the amplification of 598bp long segment and C. rectus contamination in 18 (45%) of tested samples. Mean depth of gingival sulcus in women was more but the differences in frequency of bacterial contamination between men and women and mean depth of gingival sulcus among different age groups were not statistically significant. Conclusion: The finding of this survey confirmed the presence of C. rectus in remarkable percentage of the periodontitis cases and pointed to the suitability of the PCR as an easy and rapid method for detection of this bacterium in clinical samples. Keywords: Campylobacter rectus, Periodontities, PCR

Abstract Endostatin suppresses growth and progression of many tumors through binding to endothelial cell surface and extracellular matrix proteins like integrin, heparin, matrix metalloproteinase-2 and transglutaminase-2. There is an arginine rich motif on the surface of endostatin that is essential for binding to some of aforementioned proteins. It has been shown that a 27 amino acid peptide derived from amino terminal of endostatin responsible for its anti-angiogenic and anti-tumor activities and mutation of histidines bound to Zn significantly reduce its activity. In the present study, as regards the importance of Zn-binding loop in amino terminal and arginine 27 in carboxyl terminal, peptides corresponding to this region and a mutated variant including isoleusin 26 to arginine mutation synthesized and their structure and interaction with matrix metalloproteinase-2 and transglutaminase-2 analyzed using fluorescence spectroscopy, molecular dynamic and docking simulation techniques. This study aimed to analyze effect of placing two positively charged arginines on the structure and interaction of this fragment of endostatin. Results showed that placing two arginines close together in the carboxyl terminal of peptide increases fluctuations in total structure of peptide, alters Zn-binding loop in the amino terminal and makes binding energy of peptide to matrix metalloproteinase-2 and transglutaminase-2 more negative. It can be inferred that repulsion of two positively charged arginines in carboxyl terminal induces conformational changes in the whole structure and in the amino terminal loop region.

Abstract According to the novel achievements, nanotopography and steric geometry of the microenvironment around the cells have a drastic role on their fates. Hence, fabrication of biocompatible nanostructures as the scaffolds for the cell culture and in the next step, accurate determination of their physical and geometrical characteristics is widely considered. Despite of broad utilization of Atomic Force Microscopy to investigate topological traits of sophisticated nanopatterns; its capability to characterize electrospun nanofibers has not been studied inquiringly. In the present research, chitosan nanofibers which were successfully electrospun at the optimized conditions were then evaluated using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) respectively. The results suggested that recruitment of both of these techniques have their own advantages and disadvantages. As the first noticeable issue, while the sample preparation and scanning procedure in SEM imaging may disrupt native structure of fibers, probing the sample by AFM doesn't need any pre-imaging treatment. The main application of SEM in analysis of nanofibrillar structures is the rapid survey of nanofibers shape, orientation, diameter and consistency. In the other side, three dimensional imaging by AFM makes it possible to determine whole surface roughness, roughness along fibers and woven tissue thickness. Furthermore, regarding some technical advices, AFM can be used to estimate nanofibers average diameter as well as SEM.

Abstract Human activin A is a homodimer of βA subunit which is synthesized in the form of prepro-activin with 426 amino acids; mature activin A with 116 amino acids is processed from this larger precursor protein. This protein which was extracted for the first time from follicular fluid is a strong stimulator of FSH biosynthesis. The functions have been found to be exerted by activin, including roles in cell proliferation, differentiation, apoptosis and survival of neurons. As this protein plays a considerable role in the treatment of neurodegenerative disease such as Alzheimer,s disease and wound repair, in this study for the first time was expressed in three different strains of E.coli. Activin A has disulfide bonds in its native and functional structure, so the cytoplasmic reducing environment of E.coli is not appropriate for its expression. Therefore, the oxidative space of periplasm for production of correctly folded activin A was considered. In this study, h-activin A cDNA and modified Iranian Bacillus Licheniformis α-amylase signal peptide obtained from NCBI data bank after codon optimization was cloned in pET21b(+) vector and transformed to BL21(DE3)pLysS, BL21(DE3)Rosetta gami and BL21(DE3) strains of E.coli. Expression occurred via induction of promoter with IPTG. Consequently, extracted proteins from these three strains were compared with each other using SDS-PAGE, Dot blot and western blot techniques. The data shows activin A expression especially in BL21(DE3) and BL21(DE3)Rosetta gami strains of E.coli.

Abstract Cellulase enzyme has shown their potential application in different industry. cellulase immobilization is one of the different methods for enzymatic stabilization. An advantage of immobilization is enzymatic reusability, which have an economical advantage for enzyme using in industry. Properties of Chitosan as a support for enzyme immobilization are always considerable. Due to its unique biological properties such as biocompability, biodegradability and non-toxicity, chitosan is an attractive support for immobilization. In this investigation Aa-cel9A endoglucanase gene was cloned in pET28 (+) expression vector. Sequencing result had been proved gene cloning in vector. Then the constructed vector was transformed to Eshershia.Coli (BL21) cells and enzyme production was induced. The result obtained from SDS-PAGE analysis and enzymatic assay showed the recombinant protein has been expressed and protein purification was done with Ni-NTA column. Chitosan macrobeads were prepared by precipitation procedure. After immobilization of enzyme with glutaraldehyde as linker, enzyme immobilization has been proved with FTIR and Bradford analysis. The obtained result showed optimum condition for covalent immobization on support are 0.7% of glutaraldehyde concentration and sodium phosphate buffer with pH 7. Bradford analysis and enzymatic activity assay have proved 85% of enzyme molecules immobilized on support.

Abstract This study aimed to isolate the thermophilic bacteria producing exoglucanase from hot springs of Dehloran, in Ilam province, in South-West of Iran. After sampling, bacterial enrichment was performed in a medium containing rice barn or CMC (carboxymethyl cellulose). Identifying bacteria was performed based on characteristics such as universal 16SrRNA gene sequencing using PCR. The isolates indicated the most similarity to species Pseudoxanthomonas mexicana، Chelatococcus daeguensis,Promicromonospora sp., Isoptericola variabilis sp. in the GenBank. The selected strain was identified and named as Isoptericola variabilis sp. IDAH9. The strain showed 1 U/ml exoglucanas activity. In order to optimization of enzyme production, the effects of carbon, nitrogen, Tween-80, and sucrose were evaluated. The results showed that the most exoglucanas activity are obtained at concentrations of 0.2% sucrose, 0.6% Tween 80 and 12 g/l of bran and carboxymethyl cellulose carbon sources and 5.6 g/l of ammonium sulfate. The residual enzyme activity was retained 64% of activity after 24 hour incubation at 50 °C. Therefore, Isoptericola variabilis sp. IDAH9 can be a suitable option for production thermostable exoglucanase from low price carbon sources..

Abstract Salmonella is the serious and prevalent bacteria that has important role in epidemic infections. Beside increase of antibiotics resistance in bacteria make that abundance researches did for introduction of replacement method for beard with bacteria infections. Copper NANO oxide particles are components that their anti-microbial nature be evidenced. In this research for discover of NANO particles probably mechanism on the genome of bacteria, salmonella selected as a model for Gram-negative bacteria. In this regard, at first the bacteria were treated with 30 and 60 µg/ml copper oxide NANO particles. At time intervals of 2, 4, and 24 hours. In this doses, bacteria was growth .So bacteria were treated with 90 and 120 µg/ml copper oxide NANO particles. In these doses growth of bacteria even after 24 h completely were stopped .Then their DNA were extracted. In order to investigate the effects of copper oxide NANO particles on the genome, the chain reaction techniques of (RAPD-PCR) was employed .Using the software NTSYS-PC, the results obtained from electrophoresis of PCR products on agarose gel were analysis. The results of the study revealed that copper oxide NANO particles not only affects the growth of bacteria but also affect the sequencing of genomic DNA and leads to the changes of them in different points.

Abstract Neurotrophins are a family of secretive growth factors that do their functions via binding to their specific receptors (Trks) or their common receptor (p75ntr). p75ntr has important roles in survival, differentiation and proliferation of several types of cells. MicroRNAs are non-coding small RNAs that regulate post-transcriptional mRNA expression. Recently, a MiRNA named hsa-miR-6165 has been discovered in forth intron of p75ntr. Bioinformatics analysis has revealed that this MiRNA has important roles on regulation of several cellular signaling pathways and signaling pathway involve in differentiation. Therefore, in the present study, the expression of hsa-miR-6165 in neuronal differentiation of NT2 human embryonic carcinoma stem cell line and non-nervous and nervous human cell lines was analyzed. Our results indicated that hsa-miR-6165 not only has been expressed in differentiation process of NT2 cells and neural cell lines but although significantly expressed in several human non-neural cell lines such as hFSF and Hela. In addition, the expression of this miRNA, unlike its host gene, upregulated at the end of the differentiation. These results indicate the probable presence of independent promoter for this MiRNA and revealed that hsa-mir-6165 maybe has roles in cellular differentiation which needs more investigation.