فصلنامه تازه های بیوتکنولوژی سلولی مولکولی
پیاپی 14 (بهار 1393)

Yeast expression systems have produced recombinant proteins for more than 20 years having proven to be efficient in production of pharmaceutical metabolites. The methylotrophic yeast Pichia pastoris has been widely reported as a suitable host cell for expression of recombinant proteins which has been dramatically used in recent years in pharmaceutical industry. Expression system of Pichia Pastoris has more advantages including easy genetic manipulation, high cell densities than mammalian cells, high potential for the production of recombinant protein with post-translational modifications, strong promoters for expression of recombinant protein and transformation of DNA external containing multiple copies of a target protein through homologous recombination process that they can be efficient for wide application in industrial fields. In this review, we investigated the system characteristics of Pichia pastoris yeast through genetic engineering, protein chemistry and molecular design that are necessary for the expression of the target proteins.

Aim and The hESCs can differentiate in vitro into spontaneously contracting cardiomyocytes and produce a model to investigate the early developmental stages of this system. Cardiomyocytes derived from human embryonic stem cells (hESCs) could be useful in restoring heart function after myocardial infarction or in heart failure. In this study, three cardiomyocite genes as endothelial cellular markers in vascular system were investigated. After stem cell culture, the expression pattern ANF،MLC-2a and MLC-2vwere analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). There was an enhanced expression of as myosin light chain atrial (MLC-2a) from day 15 and day 20 in EB. The myosin light chain ventricular (MLC-2v) and (ANF), atrial natriuretic gene expression were significantly increased sequentially by day45 and 20. hESCs might be useful as an effective model system for understanding the developmental processes and functioning of the human heart.

Aim and ongoing increase of world population in the world has become a complicated issue today and a crisis for future. Also, evidence showed increasing rate of suitable and available plants that have anti-productivity and adjusting potentials on men productivity. According to ingredients of nettle and their wide usage, there is little information about influence of nettle extraction on testis. Therefore, in current study, the influence of hydro-alcoholic extraction of nettle plant (Urtica dioica) on pituitary and gonadal axis that is spermatogenesis and testosterone, LH, FSH, hormones and its tissues were studied. 40 male adult rats by the weights of almost 195±5 g were divided into 5 groups each containing eight members. Rats of experimental group received 25, 50, 75 mk value of nettle extraction interior peritoneal in 28 days, respectively. The evidence group just received the soluble form of drug) distilled water while the control group just were given food and water. All groups had blood taking process on the day of 28th. Collected blood samples were used to measure concentration values testosterone, LH, and FSH hormones. Thus, tissue intersect was provided by testis. Tissue differences among experimental and control group were also studied Obtaining results evaluated by one-way ANOVA and- t-test using analysis variance analysis. result obtained showed that 75 mm /kg of nettle hydro alcohol extraction, the testosterone level of plasma in comparison with control group in statistical rate p<0.05 decrease significantly. LH and FSH hormone levels between experimental and control group showed insignificant differences. Study of histology intersection showed that sperm rate, spermatogony cell number, initial spermatosit, spermatid, and lidig in experimental group that received maximum extraction had more significant decrease than the rate of control group. Also, weights of body and testis in groups receiving 75 and 50 mg /kg of nettle hydro alcoholic extraction showed significant decrease in experimental group than control group in statistical rate of p<0.05. Due to the obtained results and other researches probably nettle extraction might have anti-androgen properties because of containing fitostrol and fitosrogen compounds and also decrease testosterone rate. Histologically, morphological modification in tubuls, inter layers destructive cells, and spermatogenesis epitilium loss were observed.

Aim and Formulation of anticancer drugs aroused intensive research in the past decades. Pegylated nanoliposomes have been utilized as effective drug carriers, a vehicle for drug delivery. The aim of this study is to evaluate the performance of polyethylene glycol on the liposome structure. In order to produce pegylated nanoliposomal certain proportions of phosphatidylcholine, cholesterol, polyethylene glycol and paclitaxel were mixed together. The mean diameter of pegylated nanoliposomal paclitaxel was determined by Zeta sizer system. The encapsulation efficiency was calculated using standard curve. The percent of drug released from pegylated nanoliposomes was performed by dialysis for 28 hours. Also, cytotoxicity of pegylated nanoliposomal paclitaxel was studied through MTT technique. The particles’ mean diameter confirmed in nano dimensions. Encapsulation efficiency and drug release of pegylated nanoliposomal paclitaxel was 95.2±6.3% and 5.02, respectively. Results showed an increase in toxicity, the formulation was prepared. Polyethylene glycol causes more stability and slower release of drug.

Aim and Cancer is an important issue in modern medicine and it is the most common cause of death after cardiovascular diseases. Breast cancer is the most common type of cancer in women and is about the cause of 1.5% of the deaths. Chemotherapy drugs, which from one side are used for prevention of uncontrolled proliferation of cells in certain tissues of body and from the other side, are used to induce apoptosis in tumor cells are important candidates for the treatment of cancer. The used synthesized compounds were derivatives of 4H-chromene 3-carbonitrile which were effective on cancerous cells resistant to the other drugs such as Paclitaxel. With respect to their ability in induction of apoptosis these compounds could be used as the therapeutic agents against cancer. Thus, the growth inhibitory activities and inducing apoptosis of synthesized 4H-chromene 3-carbonitrile were determined. Cytotoxicity effect of the synthesized4H-chromene3-carbonitrile compounds was determined against T47D breast cancer cell line using an in vitro cell culture system (MTT assay). Finally, to assess apoptosis induction of these compounds, staining methods with Acridine orange-Ethidium bromide and Hokhest staining by fluorescence microscopy and DNA fragmentation by diphenylamine method were used. The results showed that the synthesized compounds, contained two NH2 chemical groups or OH group on their benzene rings, had better cytotoxicity effect and the effect of induction of apoptosis was studied with the best compound. The cytotoxicity effect of the synthesized 4H-chromene 3-carbonitrile compounds was changed by replacement of NO2 chemical group on thiol ring with different chemical groups on the benzene ring. Therefore, the synthesized compound contained two NH2 chemical groups, had stronger cytotoxicity effect and was used to study the effect of inducing apoptosis that showed good results.

Aim& Caspian Sea is one of the most valuable ecosystems in the world containing a high diversity of fungal species with are capable of producing the enzyme. Phytases are the most important enzymes with vast applications in industry. Phytase hydrolyzes phytic acid releasing inorganic phosphate which could resolve some of the problems caused by phytate in animal feed. The aim of this study was the isolation and identification of phytase-producing fungal from the coastal waters of the Caspian Sea. The water samples were collected from west coast of the Caspian Sea in spring 2011. The samples were cultured on Potato dextrose agar and isolates were cultured on PSM agar to identify phytase-producing fungal again. After DNA extraction, phytase producing fungal were identified via ITS-5.8S rDNA amplification and sequencing. Six phytase-producing fungal species were identified from coastal waters of the Caspian Sea. Among them, three species of Aspergillus ochraceus, Aspergillus flavus and Penicillium olsonii showed higher phytase activity. Also, the two new species Discostromatri cellulare and Cladosporium gossypiicola with accession number of JF811913 and JF811912 were verified and recorded in Genebank, respectively. However, The six species were the plant pathogens fungal which entered from environment to sea and likely to have a suitable phytase activity on phytate. Due to the pollution of the Caspian Sea caused by the phytic acids and their inhibitory effect on animal food chain it can be used for bioremediation and their enzyme for processing vegetable phosphorus.

Aim and The aim of this study was identification and isolation of thermophilic bacteria from Dalaky hot spring located in Boushehr province by using of biochemical and molecular methods and considering of enzymatic properties, anti-bacterial and anti-fungal of isolated strains. Sampling from surface water, sludge and depth of one meter was carried out in sterile conditions.Samples were incubated for a week, then grown Bacteria were purified. Identification was done by using of biochemical and molecular methods. Antibacterial properties against Eshershia coli bacteria and Staphilococcus aureus by turbidometery method and antifungal properties against Aspergillus niger based on the power of inhibitory were studied. 12 strains (A1-A12) were isolated and purified. A1, A8, A9, A11 strains, can tolerate 20% sodium chloride salt. A1 and A11 strains are capable of growth at pH 3-12 and at temperatures of 20-56 ° C.The results of antibacterial effect against S. aureus strains showed that with increasing concentration of strain extract from % 5 to 20% in A5, A6, A7, A10, A11, A9, A12 strains, the antibacterial properties increased significantly. while just A12 strain prevented the growth of E-coli in concentration of 20%.The most effective strains against Aspergillus were A2 and A6. As A11 strains can grow in a wide range of temperature, pH, as well as the superior ability of antagonistic 16SrDNA coding region was sequenced. According to this A11 strain with 98% similarity was identified as Bacillus subtilis subsp. spizizenii strain NRRL. Because of anti-fungal and anti-bacterial effect, thermophilic bacteria is an excellent option in the treatment of bacterial diseases,in future studies.

Aim and Enzyme action at high salt concentrations is of practical relevance in a number of application areas. Both activity and stability of the horse liver alcohol dehydrogenase correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients (B+ for cations and B− for anions). In this study thermal stability and activity of the enzyme were invetigated. In this study, the effects of high concentrations (1- 4 M) of NaCl, NaNO3, Na2SO4 NaClO4, NaAc and concentrations (1-3 M) of KCl, NH4Cl, CsCl on the activity and stability of the horse liver alcohol dehydrogenase enzyme (HLADH) were investigated. It was found that both enzyme activity and stability of the enzyme are related to the (B−− B+) values of the salts. In the presence of salt whose anions and cations have similar kosmotropic/chaotropic properties (NaAc and NaCl), catalytic activity of enzyme showed the best conditions. kosmotropic/chaotropic properties of both cation and anion affect the catalytic activity of the enzyme by impact on the active site and catalytic mechanism and surface pH. Anions compared with cations had a more predominant role in the stability of the enzyme. Acetate anion may induce greater thermal stability (half-life increased from 300 to 560 min at 60 °C). The present work has demonstrated explicitly the impact of inorganic salts and ions on both the activity and stability of horse liver alcohol dehydrogenase. The stability study of alcohol dehydrogenase offers another example of more kosmotropic anions and chaotropic cations favoring higher enzyme stability.

Background and Biosurfactants are amphiphilic biological compounds produced extracellularly or as part of the cell membranes by a variety of microorganisms. The aim of this study was to identify a strain of bacteria of the genus Acinetobacter.spp biosurfactant producers. In this study, different samples of oil, water and soil contaminated with oil were prepared. Hemolytic activity, emulsification activity and measurement of surface tension were used and selected strains were identified by biochemical tests. The nature and effect of bacterial biosurfactant was evaluated for strain selection. In this study, eighty eight bacterial strains were isolated. Twenty four strains have been isolated from the isolated strains with hemolytic activity, among which 12 strains with high emulsification activity of 70% were separated. Finally, 4 strains were able to reach the surface tension to less than 40mN/m. On the basis of biochemical tests, a strain selected for this study were identified as Acinetobacter ssp The nature of the biosurfactants were determined by TLC, kind of the glycolipid. Furthermore, the produced biosurfactant of the selected strain had antibacterial activity against six infectious bacteria. The most sensitive ones to the effects of bacterial biosurfactant extract of Acinetobacter ssp, were Staphylococcus aureus and the most resistant bacteria to extract were Proteus mirabilis, the results of MIC, MBC showed that Extract dilution MIC at 63 and MBC Extract in concentration of 125 mg/ ml on Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa were the most effective. The findings indicated that the bacterial strains with over 70% of emulsification and the surface tension below 40 mN/m, are able to produce biosurfactant and oil degradation having stronger antibacterial effects. According to these results it can be said that this bacterium has a great potential for applications in biotechnology and environmental requirements.

Aim and Common carp is one of the most important and economical species.Using artificial reproduction method, common carp is reproduced, this method can reduce genetic diversity. In the present study the genetic structure of cultured carp in Khuzestanwere studiedusing microsatellite markers. 30 specimens of common carp were collected from a farm and Shahid Maleki Reproduction Center in May2011. DNA was extracted from caudal fin tissue by phenol-choloroform-amylalcohol. PCR was performed using 4 microsatellite markers, after survey the quality and quantity of DNA by using spectrophotometer and agarose gel electrophoresis. Ho& He ranged from 0 to 1 & 0 to 0.825 respectively. Maximum number of actual allele belonged to Shahid Maleki Reproduction Center. Half of loci showed significant deviation from Hardy-Weinberg equilibrium, mostly due to the increase in heterozygosity. UPGMA cluster analysis showed there are two different populations. The results showed maximum amount of genetic diversity belonged to ShahidMaleki Reproduction Center.

Aim and High efficient transformation of external DNA into the bacterial cells accounts as the main step of microbial biotechnology. For this purpose, some conventional methods have been developed. Using cold CaCl2 is one of the most important and cheapest ways for DNA transformation into the gram negative bacteria such as Escherichia coli. Commonly used methods have a low transformation rate about 105 to 107 transformants per 1μg of DNA. Due to the importance of DNA transformation procedure, numerous attempts have been applied to optimize the conventional methods such as cold CaCl2. In this study we showed that it is possible to increase DNA transformation efficiency into the E. coli in standard CaCl2 method, using a membrane permeable cationic peptide (CM11). Based on ClCa2 competent cell preparation method, the bacterial cells were treated by different concentration of peptide (0.5, 1, 2, 3 and 6 μg/ml) in two different ways. Thereafter pET-28a (+), pGEX4T-1 and pUC19 as model plasmids were separately transformed into the competent cells. Results showed that the highest plasmid transformation efficiency obtained at the present of 1 μg/ml of peptide. In this concentration, the transformation efficiency of pET-28a (+), pGEX4T-1 and pUC19 plasmids were respectively 4.4, 4.7 and 4 fold higher than control sample. The results of this study revealed that in the chemical cold CaCl2 strategy for DNA transformation, CM11 as a cell membrane permeable peptide can increase the efficiency of DNA transformation into E. coli.

Mycoplasma infections are contagious infections in the ostrich. At the beginning, ostrich Mycoplasma causes disease in the respiratory which have symptoms such as inflammation of the nose, trachea; also lung damage can be related to this disease. In this research, the isolation of Mycoplasma synoviae infection of the lungs and comparison of two methods (culture and multiple chain reactions (PCR)) were examined to confirm Mycoplasma contamination of ostrich lung. A multiple random samples of different parts of lung were collected from a Kerman ostrich slaughter-house; these samples were immediately collected after the slaughter in the first six months of 1391. Samples in parallel conditions were used for two methods (culture and PCR methods). The PCR product of the 16S rRNA gene was observed by UV. The egg shape of colonies and positive culture of Mycoplasma were studied on PPLO Agar culture. In addition, the molecular results of PCR product were observed on agarose gel. Although, the past research has shown that contamination in poultry flocks in industrialized regions in the country is much more than the rate of ostrich farms in Kerman but ostrich farms could serve as a reservoir for bacterial infection to spread among industrial zones of country’s poultries, despite all preventive measures.

Aim and Plant based systems offer the potential for safe, economical and high-capacity production for many biopharmaceuticals. Potato (Solanum tuberosum L.) is a proper bioreactor and a very good candidate for genetic manipulation. After gene transformation to the explants, shoot regeneration is the first and the most remarkable necessity. After investigation of various one and two-step culture media for shoot regeneration of Desiree, Agria and Marfona internodal explants and calculation of shoot regeneration percentage, the best one and two-step shoot regeneration media is introduced for adventitious organogenesis in each of these varieties. Human proinsulin gene is transferred into nuclear genome of potato plant via Agrobacterium tumefaciens-mediated transformation method. The appropriate amounts of Zeatin Riboside, Naphthalene Acetic Acid and Gibberellic Acid hormones needed for maximum regeneration in two-step culture media for shoot regeneration of Desiree, Agria and Marfona internodal explants in the first stage were (3, 0.1, 0.02), (3, 0.1, 0.02) and (3.5, 0.1, 0.02) mg L-1 respectively. These amounts in one- step culture media were (4, 0.02, 0.05), (3, 0.02, 0.05) and (4, 0.02, 0.05) mg L-1 in that order. In the second stage of two- step culture media, zeatin riboside and naphthalene acetic acid levels were reduced by 20% and the factor of 10 respectively. Assessment of transgenic plants was carried out in 3 levels (DNA, RNA and protein). The optimization of adventitious organogenesis in Desiree, Agria and Marfona cultivars will be helpful for other researchers who want to study the transformation of these cultivars in future.