NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL
Year:2015 | Volume:5 | Issue:19|Papers Count:14

Aim and Background: Among more than 23, 000 microbial secondary metabolites discovered, 42% of them are separated from actinobacteria. These secondary metabolites have wide range of biological activities such as antibacterial, antifungal, antiparasitic, antiviral, antitumor, immunosuppressive, insecticide, anti-inflammatory, antioxidant and other activities. In the past few years, numerous studies have been conducted on these biological activities in the country, but no report has been published about the antioxidant activity of local actinomycetes resources. The purpose of this research is to achieve natural antioxidants with lower physiological hazards associated with local microbial resources.Materials and Methods: Fifty strains of nativeactinomycetes preserved at the University of Tehran Microorganism collection were selected randomly and after sporolation and prefermentation and fermentation, bacterial extracts were prepared. At first, antioxidant activity of the extracts was evaluated by DPPH method. These results were evaluated with synthetic antioxidants including ascorbic acid and BHA. Also cytotoxic activity of the extracts was measured on Artemia. Elected strains were recognized with molocular method.Results: According to the results, 10% of extracts have no antioxidant activity, 20% of the extracts have highantioxidant activity (IC50<200), 32% of extracts with medium antioxidant activity (200<IC50<400), 8% of the extracts with low antioxidant activity (400<IC50<600) and 30% extracts have very low antioxidant activity (600<IC50), respectively. In the cytotoxicity test, 50% of actinomycete extracts have not lethal activity, 30% of the extracts have low toxicity, 16% of the extracts showed high toxicity and 4% of the extracts have very high toxicity.16Sr DNA analysis shows that the three elected strains are belonging to the genus Streptomyces.Conclusion: In the present study, we evaluated antioxidant activity and cytotoxicity of fifty extracts. Two strains including UTMC1054-UTMC1166 have similar power antioxidant, and lower toxicity than BHA.The extracts also showed higher antioxidant activity than ascorbic acid. Thus there is hope to achieve effective antioxidant compounds that have not toxic effects among local actinomycetes.

Aims and Background: Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that stimulates activation of mature blood cells. Nowadays, recombinant hG-CSF is successfully used for the treatment of neutropenia in patients suffering from cancer of myeloid suppressor, bone marrow transplant, acute myeloid leukemia and acute/severe neutropenia. The aim of this study was the increased expression of hG-CSF and production of the soluble protein with disulfide bonds by E.coli strains (Origami and BL21).Materials and Methods: The synthesized hG-CSF gene (in pGEM vector) was subcloned into pET23a expression vector and then the recombinant plasmid was transformed into Origami and BL21 hosts. Afterwards, expression levels were compared in two hosts.Results: Enzymatic digestion results showed the accuracy of the cloning procedures in vector pET23a. Also under the same conditions and after induction of different hosts with IPTG, best hG-CSF protein expression was observed in strain Origami E.coli.Conclusion: The results showed that E.coli (Origami) is appropriate strain for the expression of human G-CSF and this strain can be used as suitable host for the expression of this protein in higher scale.

Aim and background: lipase is a carboxylic ester hydrolase enzyme (EC.3.1.1.3), which acts on triglycerides to release fatty acids, monoglycerides, diglycerides and glycerol. With respect to the biological process advantages against chemical process plus the ability of lipase to catalyze the revers reaction (i.e., esterification) a vast chapter on this enzyme scientific applications was started. The focusing point in this study was to determine the optimum cultivation time for free and immobilized form of Rhizopus oryzae with regard to producing membrane-bound lipase. With the usage of n-butanol and oleic acid in a non-polar environment (n-hexane as solvent) the esterification reaction was carried out while the effect of molecular sieves on the final yield was.considered Materials and methods: Rhizopus oryzae cells in the form of immobilized on cellulose support particles or free cells are used as a whole cell bicatalyst. Colorimetric method was used for measuring the esterification activity (i.e., determination of residual free fatty acid in reaction.) media Results: Membrane-bound lipasees which are produced by both, immobilized and free form of the cell reach their maximum activity after 48 hour. Also the biocatalyst esterification activity was improved approximately 4-folds by the immobilization of fungal cell on loofa sponge. More than 96% efficiency was gained in synthesis of 1-butyl-oleate ester in the presence of 4 g. molecular sieve Conclusion: Immobilization could improve the membrane-bound lipase esterification activity. The lipase secretion to the culture media significantly affected by cultivation time. To reach a complete esterification reaction (approximately 100%), water adsorption with use of molecular. sieve is an appropriate method.

Aim and Background: In recent decades, drug carriers in nano scale have been focus of attentions. In last two decades, cyanoacrylate nanoparticles have been widely studied as drug nanocarriers. In this work, production of polybutyl cyanoacrylate (PBCA) nanoparticles by emulsion polymerization has been investigated.Material and Methods: polybutyl cyanoacrylate nanoparticles were produced by emulsion polymerization using 70 KDa dextran as stabilizer. Nanoparticles were described by Zeta sizer, scanning electron microscope and light microscope. Effect of polymerization factors on size and distribution of particles, was studied. Such factors include 70 KDa dextran, polysorbate-80, concentration of H+ion, polymerization time, temperature and sonication.Results: This study indicates that a definite portion of stabilizer is essential for the production of nanoparticles. This concentration was recognized to be 2%. Effect of H+ion was considerable as a sharp decrease was observed in pH 4. In comparison with room temperature, higher size and distribution of nanoparticles was gained in temperature of 50 °C. Effect of polysorbate-80 in concentration of 0.001% on nanoparticles was evaluated to be positive. Furthermore, increasing polymerization time from 5.5 to 18 hours resulted in synthesis of more appropriate nanoparticles.Discussion: The results illustrate that nanoparticles properties depend on various factors and manipulation of such factors can result in desirable properties.Conclusion: PBCA nanoparticles are under the influence of various factors and we can make an acceptable and desirable nanoparticle by their proper manipulation.

Aims and Background: RNA extraction with high quantity and quality is a fundamental requirement for molecular biology studies. Presence of carbohydrate, tannins, proteins and polyphenols compounds have created problem for extracted RNA from woody plant. Therefore, it is important to a suitable extraction method to produce a good quality RNA.Materials and Methods: Methods for RNA extraction were compared to choose the best method of RNA extraction from pomegranate (Punica granatum L.) peel. These methods included 1-Zarei et al 2- IHTB 3-CTAB-LiCl modified method.Results: Of the methods tested, Zarei et al method is the most appropriate method for plants with high phenolic compounds, especially pomegranate. Hence the need for the use of dangerous chemical material and extraction kits is not felt.Conclusion: At the end of experiment with attention to results and comparison between methods, Zarei et al method was selected as the best method for RNA extraction from fruit peel with high quantity, quality and purity. Maximum amount of RNA with high quality that extracted from Zarei et al method was 642 ng per 100 mg peel tissue.

Aims and Background Nanotechnology is used to design new formulations of drugs. Also, nanocarriers are utilized fordrugs or biologically active substances to improve the therapeutic index. Chemotherapy effectiveness has direct proportion to increase the time of exposing cells near drug. Due to their small size the nanocarriers will then penetrate into the target tissue causing an efficient drug accumulation at the target position.6-gingerol which inhibited efficacy on cancer cell growth in this research preparation as formulation of nanoarchaeosomal PEGylated 6-gingerol and evaluate its impact on cancer cells MCF-7 cell line.Material and methods FirstHalobacterium salinarum lipids were extracted and added 6-gingerol (dissolved in ethanol), Polyethylene glycol (PEG) 2000 and Twee n 80 to it and with the use of Avaporator extract the organic solvent, then were dissolved in buffer phosphate environment and Sonication. Their particle diameter measured with Zeta sizer device and finally evaluated the effect of the formulation cytotoxicity by MTT.Results The diameter of the nanoarchaeosomal PEGylated 6-gingerol was equal to 290 nm. The encapsulationefficiency of 6–gingerol in nanoarchaeosomal PEGylated was also equivalent to 96/33 percent, respectively. The Cancer cells against the effect of this formulations showed difference in different doses.Conclusion The dimensions of the nanoparticles and the encapsulation efficiency was suitable for targeted drugdelivery, and nanoarchaeosoma PEGylated 6-ginger showed cytotoxicity effects on breast cancer cells MCF-7 cell line.

Aims and Background: Cellulose is the most widely used biopolymer to produce paper, textiles and biomedical materials. One of the interesting methods to produce cellulose is microbial resource due to the shortage of cellulose original resources. On the other hand, the use of microbial cellulose as a pure polymer and absence of lignin and hemicellulose is preferred. The present study was aimed to isolate Pseudomonas luteola producing cellulose bacterium to produce cellulose and evaluate its production quality.Materials and Methods: Samples from different local vinegars, juices and fruits were cultured in Hestrin-Schramm agar medium. Pseudomonas luteola was isolated with biochemical tests and verified by 16S rRNA typing method. After cultivation of the isolate in Hestrin-Schramm broth, cellulose was produced in medium. The cellulose layer was treated by SDS and NaOH, then purified and dried. Finaly, the produced cellulose was verified by cellulase enzyme and scaning electron microscope (SEM).Results: In this study, the gram-negative organism Pseudomonas luteola was isolated and identified. Then, the isolate was cultured in Hestrin-Schramm broth and cellulose was produced after 6 days incubation at 30oC. The enzymatic digestion results were showed that the cellulose has been presented in culture medium. Scaning Electron Microscope (SEM) image was revealed that the produced cellulose by the isolate has fibrous and plexiform structure.native isolate of Pseudomonas luteolaConclusion: This is the first report on cellulose production by in Iran. The quality of produced cellulose by the organism is appropriate in comparison with the cellulose produced by standard strain, thus is presented to produce cellulose as a new resource.

Aim and Background: Biosurfactants or Bioemulsifiers are surface tension-reducing compounds that are produced by many bacteria and fungi. Importance of these compounds in comparison with synthetic surfactants can be outlined as no toxicity, environmental friendly, activity in high temperature, pH and salinity. The purpose of this research was the isolation and identification of biosurfactant-producing Lactobacillus and evaluating the effectiveness of produced biosurfactant.Materials and Methods: In this study, sampling of dairy products and culturing in MRS medium was performed. The isolated strains with capability of production of biosurfactant were identified based on the biochemical and molecular methods. The optimum conditions of biosurfactant production were investigated and its emulsifier activity was studied in a food model system.Results: Altogether, nine strains of Lactobacillus isolated from the dairy products among them, Lactobacillus plantarum and Lactobacillus pentosus had the greatest capability for biosurfactant production. The extracted biosurfactants from both bacteria showed that they have good capability in emulsification process.Conclusion: The results of this research showed that it is possible to isolate strains of lactobacillus with appropriate capability of biosurfactant production and if the costs of production can be reduced, there is possibility of using such strains for production of biosurfactant in industrial scale as a substitution of synthetic emulsifiers in food industry.

Aims and Background: Nerium oleander is a medicinal plant with various biological activities such as antibacterial, antioxidant and cytotoxic effects. In the present work, methanolic extract of plant was used for the evaluation of toxicity and teratogenic effects on chicken embryo as an animal model.Materials and Methods: Methanolic extracts were prepared. Methanolic extract solutions in DMSO were injected in air sac of chicken eggs at concentrations of 5, 10, 20, 40, 60 and 80 mg/egg. Seventy two hours after incubation, eggs were placed at 37.7-38oC incubator.Results: On the day 19 after incubation, eggs were opened and shown that 78.4, 65, 56.7, 46.7, 31.66 and 21.66 percent of embryos were alive, Results showed that LD50 was at the concentration of 43.60mg/egg. Morphological and skeletal abnormalities in the treatment groups showed that club foot and beak deformity were occurred in morphology while caudal vertebrate deletions, unossification or uncalcification of caudal vertebrates were appeared in skeleton of the Chickens.Conclusion: The study of methanolic extract showed that, with increasing concentration, mortality increased. The extract in low concentrations had negligible toxicity and teratogenic effects which can be used as therapeutic aims.

Aim and Background: Dill (Anethum graveolens L.) is one of the most important medicinal plants with great applications in different medicinal industries. In present study, effects of biological and chemical nitrogen fertilizers were evaluated on growth, yield and essential oil composition of dill.Materials and Methods: This research was conducted under field condition in randomized complete block design with three replications and two factors. The treatments included Nitroxin biofertilizer in four levels (control, seed inoculation, topdress, seed inoculation and topdress), and chemical fertilizer was applied in three levels (0, 50, 100 Kg/ha), combinations of biofertilizer and chemical fertilizer and control. Different characteristics such as plant height, number of umbel per plant, number of umbellet per umbel, number of grain per umbelet, 100 seed weight, grain yield, biologic yield and oil percentage were recorded.Results: According to the results, the highest height, biologic yield and grain yield components (except harvest index) obtained on biological fertilizer. Results showed the highest biologic yield and grain yield components, essential oil percent (except the number of umbel per plant, 100 seed weight and harvest index detected in chemical fertilizer). Results showed that the highest essential oil content was detected in biological fertilizer and chemical fertilizer. Identification of essential oil composition showed that content of carvone increased with the application of biofertilizers and chemical fertilizers.Conclusion: Application of biofertilizers enhanced yield and other plant criteria in this plant. Generally, it seems that using biofertilizers or combinations of biofertilizer and chemical fertilizer could improve dill performance in addition to reduction of environmental pollution.

Aim and Background: ICD-85 with weight of 10, 000 to 30, 000 Daltons is a triple peptide derived from Iranian brown snake venom, scorpion venom of Agkistrodon Halys and yellow Hemiscorpius lepturus. This compound inhibits cancer cells by inducing apoptosis. In this study, the preparation and labeling of this triple peptide has been done.Materials and Methods: At first, these triple peptides were isolated from snake and yellow scorpion venom by gel and high performance liquid chromatography. Labeling with iodine-131 was performed through chloramine-T method and radiochemical analysis involved sephadex G-25 method. The stability and biodistribution of radio peptide was checked in the presence of PBS and mice respectively.Results: The results of biodistribution showed that the labeled compound has about 15% accumulation in breast tumor and in other organs such as liver and kidney they have the highest radio peptide concentration. Tumor to blood ratio for radio peptide is more than 293 percent.Conclusion: The labeled ICD-85 by 131I can be an agent for diagnosis of breast cancer tumors.

Aim and Background: In this study, the efficacy of silicon (Si) was determined in order to increase the pool of phenols and antioxidant activity in plum (Prunus salicina Shablon) fruits and, thereby, enhance postharvest plum fruit quality.Materials and methods: The fruits were randomly divided in two groups so that Si-treated group immersed for thirty minutes in solutions of 5 g/l Si and were air-dried at room temperature. The activities of catalase (CAT) and superoxide dismutase (SOD) were determined.Results: Postharvest silicon application had no effect on CAT activity; in contrast, weight loss, total phenolics concentration, lipid peroxidation as well as phenylalanine ammonia-lyase (PAL) and SOD activity responded positively to the Si treatment. Silicon application raised phenylalanine ammonia-lyase activity and total phenol concentrations led to increase the fruit’s ability to better withstand stressful cold impacts. In addition, Si considerably reduced the rise in malondialdehyde (MDA) content during storage, obviously because of an efficient scavenging following significant enhancement of SOD activity.Conclusion: Postharvest Si application raised antioxodant activity and total phenolic, reduced malondialdehyde content and lipid peroxidation, and led to increase the fruit’s ability to better withstand stressful cold impacts. These results suggest that Si application may be recommended for increasing storage life and maintaining their quality.

Aim and background: Archaeosome nanoparticles are used in drug delivery to improve the efficacy of therapeutic agents and decrease the side effects. In this study, archaeosome nanoparticles were used as Cisplatin carrier.Material and methods: Methanogenic archaea were successfully cultured in a routine liquid medium. Archaeosome was extracted from the membrane of archaea using centrifugation process. After that homogenous emulsion of archaeosome was raised in phosphate buffer saline containing poly ethylene glycol and Cisplatin under motion by stirrer. Sonication performance was also recruited to obtain the nano sized archaeosome and Cisplatin entrapment. While Cisplatin encapsulation efficiency was estimated in supernatant after separation by ultracentrifuge, cytotoxicity effects of Cisplatin in the free form and encapsulated on the archaeosome were calculated in the breast cancer MCF-7 cell line using MTT assay.Results: Drug-containing nanoparticles were synthesized successfully. The entrapment efficiency for pegylated and non pegylated nano archeasomes was estimated 70.16 and 54.52% respectively. Size of the nanoparticles was shown to be 132.84 and 388.31 nm for non pegylated and pegylated nanoparticles respectively. Although cytotoxicity effects of Cisplatin was increased when drug become associated with NPs, this effect was predominant when pegylation process was contributed to construct the Cisplatin loaded nano archaeosome.Conclusion: The results of our study suggested that archaeosome nanocarriers could be used as a suitable carrier for Cisplatin while pegylation increases the efficiency of preparation.

Aim and background: Cancer/testis (CT) antigens are a category of tumor antigens that are typically expressed only in the human germ line, and in several types of tumor. MAGEA4 is one member cancer testis antigen family that has relation with other tumors. Since the biological function of MAGEA4 is unclear, the aim of the present study was amplification and cloning of the gene in the expression vector to produce recombinant protein that expresses MAGEA4.Material and method: Using PCR specific primers including restriction site, MAGEA4 was amplified. The purified PCR products were ligated between the BamH1 and Xho1 sites of pTZ57/R cloning vector and transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. The correct orientation of MAGEA4 fragment was identified by restriction enzyme analysis and sequencing of constructed plasmid. Then sub cloning was carried out within pRUF expression vector with the same restriction site.Result: The final confirmation was performed using colony PCR, double digesting and sequence analysis. Therefore the MAGEA4 gene was cloned into the restriction site of pRUF.Conclusion: This study is an important step for producing recombinant proteins and is used to find the function of this gene for therapeutic targets.