فصلنامه تازه های بیوتکنولوژی سلولی مولکولی
پیاپی 15 (تابستان 1393)

Aim and Thermostable DNA polymerase enzyme has been taken much into consideration due to its application in PCR and molecular biology researches and it has doubled the importance of the study on the various thermostable DNA polymerase. The purpose of this study was to assay the function and efficient production of the cold sensitive thermostable DNA polymerase and its quick and cheap purification. After synthesis of the designed gene artificially, it was cloned into the pET28 vector and then was transferred into E.coli. IPTG was used as an inducer in the study of gene expression. Initial purification of the enzyme was performed by heat treatment at 72 °C and precipitation of other insoluble proteins by centrifuge. The DNA synthesis activity of crude extract was compared with commercial enzyme. Recombinant cold sensitive and thermostable Taq DNA polymerase expressed in E.coli showed a significant advantage and more desirable functionality such as activity and thermostable compared to commercial enzyme. With regard to importance and application level of thermostable DNA polymerase in molecular biology, the production method used in this study is practical and cost effective. Additionally, the simplicity of producing method of this enzyme and its accuracy is a good reason for artificial and local production of highly pure cold sensitive Taq DNA polymerase.

Aim and Phylogenetic analysis and morphological identification on species of each region can be used as the basis for establishing subsequent studies. Many phylogenetic studies have been carried out according to mitochondrial genome on gastropods group in other coastal areas of the world but there are not much data on nuclear genome. Most Gastropod study in Iran is founded on morphology of the species, and there are no records of molecular study from south coast of Iran. In this study phylogenetic analysis of a sea slug species from Chabahar coast was examined for the first time. Samples were collected from rocky shores of Tis in Chabahar and transferred to the laboratory. The samples were analyzed morphologicaly prior to being frozen in freezer -80 degrees. DNA was extracted by using CTAB method and then Polymerase Chain Reaction, sequencing alignment were conducted and phylogenic tree was obtained. Nucleotides sequence of the gene in 18s rDNA region was analyzed. The phylogenetic analysis was conducted and found that the Iranian species and other species in Panpulmonata clad have more divergence than species of the other two clads. Maximum likelihood phylogenetic analysis showed that the Iranian species from Chabahar coast is in the Panpulmonata clad and with 100% bootstrap support is a sister group Peronia Cf.peroni. The molecular results are confirmed by morphological features.

Aim and Todayhydrocarboncontaminationisa majorenvironmental problem. Petroleum hydrocarbons are mix compounds divided into four groups: saturates, aromatics, resins and asphalten. Among various phase of crude-oil, alkane with mediumchain(C10-C20) are favorable substrate that are rapidlydegraded, although short chain alkanesare verytoxic whilelong chain(C20-C40)oneshave low solubility in water that reduce bioavailability andmakeresistant to biodegradation. In this research, for the isolation of aliphatic degrading bacteria samplingwas performedin petroleum reservoir waste water in Tehran, Kerman and Semnan and also soil contaminated tohydrocarbon. Alkane degrading bacteria were isolated by enrichment in Bushnell-Hass medium with hexadecane as sole source of carbon and energy. Alkane hydroxylase gene wasidentified all strain by PCR with specific primers. Inthis study,15strainswere isolated from bacterialde composition of aliphatic compounds, and only one of them, i.e. P2, did not growon thealiphaticcompounds(Hexadecane as the solecarbon source). Thismeansthere isnodegradationofhydrocarbonsbythis bacteriawill take place by this bacteria;butother strainswereable to growonhexadecaneandalkanehydroxylasegenewasconfirmed byPCR. This experimentshowsthatbacteriahave theability to degradealkanes whichmusthad alkaneshydroxylasegene,so,lack of this genecauses indissoluble of the aliphaticcompounds.

Aim and carboplatin as an anticancer agent belongs to platinum family drugs. Along with anticancer functionalities, this drug may have some side effects. Targeted and selective prescriptions of drugs reduce their side effects. Nanotechnology-based drug delivery systems are available approaches to overcome this problem. In this study, carboplatin was loaded on poly butyl cyanoacrylate nanoparticles and then its features were evaluated. carboplatin was loaded into nanoparticle during polymerization of monomer. Monomer was polymerized by emulsion polymerization technique. To determine amount of loaded drug on nanoparticle, spectrophotometery method was used. Evaluation of the cytotoxicity effects of free form of drug and carboplatin-loaded nanoparticle were performed on MCF-7 cell line using MTT assay. size and zeta potential of carboplatin-loaded nanoparticles were found to be 319 nm and -7 mv. Loading percentage of carboplatin on nanoparticles was estimated 6%. Evaluation of cytotoxicity effects shown survival rate due to carboplatin and carboplatin-loaded nanoparticle at concentration 20 macro molar (p<0.01) were estimated 80±0.6% and 84±0.6%, respectively. These values at the concentration of 160 macro molar (p<0.01) were estimated %44±0.5 and %51±0.2, respectively. Probably, due to low loading efficiency, cytotoxicity effects of carboplatin-loaded nanoparticle were decreased in comparison with free form. Further studies were needed to construct suitable carboplatin-loaded poly butylcyanoacrylate nanoparticle. In this way miniemulsion polymerization could be recruited.

Aim and Archaeal lipid derived liposomes referred to as Archaeosomes are among newly studied adjuvants with some specific immunopotent properties. In the present study large scale cultivation of Methanobrevibacter smithii, lipid extraction of obtained biomass and archaeosome production was assessed and evaluated. archaea Methanobrevibacter smithii, DSMZ 2375 was grown in the fermenter under controlled pH, temperature and anaerobic atmosphere condition to achieve archaeal cell mass. Total polar lipids were extracted via methanol/chloroform/water (2:1:0.8 v/v) solution from the frozen and thawed archaeal cells obtained from large scale cultivation. Archaeal liposomes prepared through hydration of extracted polar lipids by PBS buffer containing bovine serum albumin as the tentative antigen. Hydration was also carried out by BSA free PBS buffer to prepare antigen free archaeosomes to be compared with antigen containing type. Purity of extracted lipids evaluated via agarose gel electrophoresis and SDS-PAGE. Antigen concentration and entrapment analyzed by Lowry method and SDS-PAGE, respectively. Analysis of extracted lipids and archaeosomes revealed the high concentration and purity of obtained total polar lipids and successful entrapment of antigen in the archaeosomes. Adjuvant application of archaeosomes obtained from total polar lipids of Methanobrevibacter smithii could be a promising achievement in a variety of vaccine studies.

Aim and Influenza A is a major cause of mortality in a year. There has been an efficient vaccine available against it since 1960. Antigenic variation is a preventive agent for yearly vaccine production. Now researchers focus on the constant antigens of viruses. M2 protein makes an ionic channel in the coat of influenza, a virus that is effective on infection by virus. This protein is conserved and it is considered as a proper candidate vaccine for influenza infection. Due to limitations for influenza virus culture at the lab, the sequence of influenza virus H1N1 strain of Iran was synthesized. It was sub-cloned into pET22b vector recombinant protein and expressed in BL21 E.coli and confirmed with specific antibody by SDS-PAGE and western blot techniques. The concentration of recombinant M2 protein was 300μg/ml confirmed by specific antibody. Influenza M2 protein could be expressed in BL21 E.coli host.

Aim and Bombesin is a peptide showing high affinity for the gastrin releasing peptide receptor (GRPr). The prostate, lung, breast and colon tumors express many GRP receptors. The aim of this research was to compare two Iranian radiolabeled BBN analogs based upon the DOTA and Hynic chelators which could be used as a tool for the diagnosis of GRP receptor-positive tumors. Two peptides were synthesized using a standard Fmoc strategy which lead to produce DOTA-GABA-Bombesin (7-14) NH2 and HYNIC-GABA-Bombesin (7-14) NH2. Peptide conjugate showed good stability in the presence of human serum (37 degree centigrade). The internalization rates were studied in GRP receptor expressing PC-3 cells. Biodistribution of radiopeptide was studied in nude mice bearing PC-3 tumor. The results of internalization sowed that [67Ga]-DOTA-GABA-Bombesin (7-14) has a more internalization relative to 99mTc-HYNIC-GABA-Bombesin (7-14) NH2 in three stages. The results of biodistriution showed that the Tumor/Kidney and Tumor/Pancreas ratio after for [67Ga]-DOTA-GABA-Bombesin (7-14) was better than the other one. These data show that two bombesin analogs are specific radioligand for gastrin-releasing peptide receptor positive tumors and is a suitable candidate for clinical studies. The results of blood clearance and internalization showed that the [67Ga]-DOTA-GABA-Bombesin (7-14) has good conditions as an imaging agent relative to the other one.

Aim and Intrauterine exposure to maternal tobacco smoking is associated with increased morbidity and mortality of the human infant. Nicotine interacts with endogenous receptors in the brain and lung, and developmental exposure produces structural changes as well as alterations in neuroregulation. The present study evaluated the possible role of nicotine on the developing rat fetus that include developing brain and fetus weight. Wistar rats (250-300 g) were used in this study. After pregnancy, the animals were divided into control and nicotine (0.3 mg/kg) in tap water groups. On 19th embryonic day, the rats were killed and their embryos were surgically removed, washed, and their weight was measured and fixed in formalin 10%. Then tissue processing, cutting and Hematoxylin-Eosin (H&E) staining were preformed. The samples were evaluated using light microscope and MOTIC software for changes in hippocampus area. Data were analyzed using SPSS software version 9.01. The weight of embryos decreased in the nicotine group. In addition, embryonic hippocampus layer the treatment group was change compared with the control group. It seems that nicotine induced its influence on embryo development via different pathways and has a toxic effect on the brain.

Aim and Urinary tract infections (UTIs) are widespread health concerns which differ according to geography and regions. Bacterial Urinary tract infections associated with an enlarged antimicrobial resistant species are found in all age groups.The aim of this study was to ascertain the antimicrobial resistance pattern of strains isolated from patients with Urinary Tract Infection (UTI) and the emergence of multidrug resistant bacterial strains. Midstream urine samples were collected from 2235 patients and analyzed for isolation and identification of Multidrug Resistant (MDR) strains. The MDR strains were recognized by the Kirby Bauer method according to National Committee of Clinical Laboratory Standards. Female patients constituted 308 (62%) in the study. This result indicates that E.coli is the predominant pathogen causing UTI, followed by Klebsiella, Streptococcus viridans, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Proteus and Pseudomonas. Bacterial isolates from patients with UTI showed high levels of single and multiple antimicrobial resistances against commonly prescribed drugs. The present study confirms that bacterial resistance would be a serious problem in the country. Therefore, antimicrobial surveillance and in vitro susceptibility testing with strict adherence to antibiotic policy may facilitate to control the spreading of drug resistant microbes.

Aim and Carriers have made a big evolution in the treatment of many diseases in recent years. Lipid carriers are of special importance among carriers. Archaeosome is one of the lipid carriers. In this study, paclitaxel was archaeosomed to reduce side effects and improve its therapeutic index. In order to prepare nanoarchaeosomal paclitaxel, Archaeosomes are synthesized with a certain ratio of paclitaxel in PBS. The mean diameter of archaeosomal paclitaxel was calculated by Zeta sizer. Encapsulation efficiency of the prepared formulation was calculated by spectrophotometry. Cytotoxicity effect was determined by MTT method. Drug release pattern was determined by dialysis in 26 hours. Using artificial neural network, amount of released nanoarchaeosomal drug was predicted accurately. In this method, the release was predicted by 4 layers and 20 neurons in hidden layers. The mean diameter of archaeosomal paclitaxel was found to be about 521.4 nm. Encapsulation efficiency of the prepared formulation was 99.1±0.21. Drug releasing of archaeosomal paclitaxel was 0.149% within 26 hours. Predicted values of release for nanoarchaeosomal paclitaxel have had R= 0.99716 and MSE=4.01 × 10-13. The used model demonstrated that artificial neural network technique can predicts the amount of release with high precision. Also it is possible to predict released amount of other drugs by this model. Although drug release has special pattern which should be considered.

Aim and Helicobacter pylori has been recognized as a major risk factor for the development of gastritis, gastric and duodenal ulcers and gastric cancer. Various methods have been known to detect this microorganism and the most common method especially in patients undergoing endoscopy is rapid urease test (RUT), but this test is potential to give false positive and negative results. Therefore, using a rapid, simple but high-precision method is needed to confirm this test in laboratories. The objective of this study was to evaluate the diagnostic value of RUT compared to PCR molecular method for the diagnosis of H.pylori. This study was carried out on 100 biopsy samples collected from dyspeptic patients attending the endoscopy. The rapid urease test was performed on all samples and the PCR test was conducted with designed primers on glmM gene. Diagnostic values of RUT and PCR were evaluated using SPSS software. The product of optimized PCR with 201bp length correctly amplified and observed on electrophoreses gel. Evaluation of the selected primers with various DNA demonstrated high accuracy. Sensitivity of the test was 10 CFU with 100% specificity. In this study, 85% of specimens were detected with PCR whereas only 63% of them were detected by the RUT. In this study, it has been approved that PCR can be used for the diagnosis of H.pylori on clinical specimens because it is highly sensitive, specific and can be used on biopsy samples from RUT.

Aim& One of the chemotherapy drugs used to treat breast cancer is a paclitaxel which may be followed by side effects such as weakening of the bone marrow. It has recently been shown that nanotechnology can be used to further reduce side effects, and increase the efficiency of treatment. The aim of this investigation is to prepare nano-niosomal pegylated paclitaxel and nano-liposomal pegylated paclitaxel nanoparticles for its cytotoxic effect on breast cancer cell line. Paclitaxel by ether was prepared by injection method of nano-niosomal pegylated and nano-liposomal pegylated form. For preparation of nano-niosomal pegylated paclitaxel, specific ratio of Span 60, cholesterol, and polyethylene glycol 2000 Dalton and paclitaxel were mixed. For the preparation of nano-liposomal pegylated paclitaxel specific ratio of phosphatidylcolin, cholesterol and polyethylene glycol 2000 Da and paclitaxel were mixed. Also the dialysis bag method and MTT test were used to check the release and toxicity of formulations prepared. We were able to increase encapsulation amount of paclitaxel drug in niosomal pegylated nanoparticles with a significant amount of liposomal pegylated paclitaxel nanoparticles. Niosomal pegylated nanoparticles average diameter is smaller than the nano-liposomal pegylated paclitaxel. The encapsulation rate was significantly higher in both formulations. This study showed that the niosomal paclitaxel formulation has a better performance than liposomal paclitaxel formulation and apart from nano-carrier, both formulations have the potential of in-vivo experiments and clinical outcome are discussed. By dialysis, drug release in nano-niosome and nano-liposome Paclitaxel formulation within 48 hours was studied. This study showed that cytotoxicity effect of nano-niosome and nano-liposome Paclitaxel is more than that of standard form.

Aim and Environment is the likely source of most Non-Tuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the isolation methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. 38 samples were collected from surface waters in Tehran. Water sampling was carried out in a volume of 200-100 ml. water sample was transferred immediately to the laboratory and examined. Three methods (Cetylpyridinium chlori, Petroff, Tacquet-Tison) for the isolation of mycobacteria were compared by applying them in parallel to 38 samples of surface water. Each method was defined by a particular combination of decontamination method. The efficacy of each method was determined by calculating the positivity, negativity, and contamination rates, the mean numbers of mycobacterial colonies grown and number of different mycobacterial strains isolated. The last value was determined by subjecting the isolates to PCR. Decontamination with CPC appeared to be the best decontamination method, on the one hand, it significantly decreased the level of non-target microorganisms and, on the other hand, it was significantly less lethal for the NTM strains studied. Our goal was to measure the effects of various methods known to inhibit the growth of non-target microorganisms, while we also took into consideration the inhibitory effects of these methods on the growth of NTM. We propose that CPC procedure could be used for detection of NTM in aquatic samples.