فصلنامه تازه های بیوتکنولوژی سلولی مولکولی
پیاپی 7 (تابستان 1391)

Astrocytes are special glial cells by a population of about 10 times more than the population of the neuron within the central nervous system (CNS) as they occupy almost the 25% to 50% of the brain volume. They are apparently stellar in shape and have many processes enabling them to produce non synaptic contacts with neural cells. Using perivascular feet they are covering near to 85% of CNS capillaries external surface. Astrocytes are arranged like building blocks all over the CNS and connected to each other by Gap junctions producing an electrical syncytiym. In the previous knowledge of medicine، within the CNS and between the glial cells، microglia were considered as more important player than the Astrocytes، as Astrocytes were seen merely as guard cells and filler of empty spaces in the CNS. However studies at the last 20 years were clarified that the Astrocytes are important not only for physical support and neural nourishment but also for neural system development and synapse formation، neural tissues blood current and energy regulation، neurotransmitter adjustment and even immunological responses within the CNS. The present article is to review and point on the different roles of Astrocytes within the CNS.

Aim and Pseudomonas Aeruginosa Elastase is an important enzyme in basic studies on Zn-metalloproteases and is also considered as a virulence factor of the bacterium. In the present study، the gene encoding Pseudomonas aeruginosa elastase (PAE) was extracted and clone، Following expression and purification، its biochemical properties including optimal temperature and pH، and also enzymatic activity in the presence of glycerol، dimethylformamide (DMF)، methanol، ethanol، ethylene glycol and isopropanol were investigated. Bacterial strain was verified by biochemical tests including citrate، SIM، MR-VP and TSI. Genomic DNA was extracted using gene extraction kit and elastase gene was amplified by PCR using two specific primers. Cloning was carried out within pET21a (+) vector with HindIII and NdeI restriction sites and transformation accomplished by chemical method. The cells were cultured under the given optimized conditions، in LB medium with 1mM IPTG. Following the strain verification by biochemical tests، genomic DNA was extracted and PAE encoding gene was isolated using specific primers. Elastase gene، which is encoded as pre-proelastase، was cloned within pET21 a (+) vector and transformed into E. coli strain BL21. Nucleotide sequence analysis shows an open reading frame (ORF) of 1497 nucleotides corresponding to 497 amino acid residues. Following the induction by IPTG، the active enzyme was detected in the soluble fraction. Consequently، the enzyme was purified using affinity chromatography. Enzyme properties such as optimal temperature and pH، irreversible thermo inactivation and organic solvent activity were investigated. Our results clearly indicate that the above mentioned enzyme possesses a noticeable activity and stability within organic media. Elastase from Pseudomonas aeruginosa strain PTCC1430 shows only one amino acid substitution in the signal sequence in comparison with that of strain PAO1 whose crystal structure is solved. Biochemical properties reveal that elastase is a highly stable enzyme in organic solvents.

Aim and Cardiomyocytes derived from human embryonic stem cells (hESCs) could be useful in restoring heart function after myocardial infarction or in heart failure. The hESCs can differentiate in vitro into spontaneously contracting cardiomyocytes and produce a model to investigate the early developmental stages of this system. After removing of cells from their feeder layer، hESCs create embryoid bodies (EB). Plating EB results in developing areas of beating cells. The expression pattern of Alpha-cardiac actin and FLK1 (Vegfr-2/KDR) were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). There was an enhanced expression of as Alpha-cardiac actin from day 10 in EB in suspension. The FLK1 (Vegfr-2/KDR) gene expression was first specified in 4-day-old EB and was significantly increased by day 10 & 14. hESCs might be useful as an effective model system to understand the developmental processes and functioning of the human heart.

Aims and Amylases are one of the major industrial enzymes that occupy 25 % of the world enzyme market. These enzymes are used in many different areas، such as paper، textiles، pharmaceutical، food، starch saccharafication and processing industries. Much research has been carried out regarding application of amylolytic enzymes produced by the genus Bacillus، to the production of glucose and beverages، food and detergent industries. Because of the extensive applicability of amylolytic enzymes، research in relation to identification of these enzymes and their characteristic is continuously being carried out. Hence، the aim of this research was to investigate the amylolytic enzyme profile of a native Bacillus strain، and determine its enzyme characteristics. In this study، the enzymatic profile of the bacterium، Bacillus amyloliquefaciens BEH 111، was determined by using the SDS-PAGE، zymogram and TLC methods. Zymography and TLC were used to identify the amylolytic enzyme profile of the bacterium. The molecular weight of α-amylase was estimated by SDS-PAGE. The 3، 5-Dinitrosalisyclic acid (DNS) indicator was used to examine α-amylase activity، quantitatively. Investigation of enzyme characteristics and determination of maximal enzyme activity was carried by the DNS method، at different temperatures، pH values، substrate concentrations and time periods. The molecular weight of α-amylase was estimated to be 55 kDa by SDS-PAGE. Investigation of enzyme characteristics showed that maximum α-amylase activity was achieved at a temperature of 50oC، pH 7، presence of 2% starch، during a 90 min time period. Enzyme activity obtained under these conditions was 8222 U/L. The high level of enzyme activity produced by B. amyloliquefaciens BEH 111 can thus be of commercial significance. Furthermore، by having activity at 50oC and above، makes it possible for this enzyme to be applied to processes that use high temperatures. The optimized pH levels of 6-7 that resulted in high enzyme activity can also allow the potential use of this enzyme in processes such as starch liquefaction and gelatinization.

Aim and As mycobacterial species have different drug susceptibilities، precise identification is crucial for adoption of correct drug therapy and can ultimately influence patient outcome. Among various molecular methods، PCR-restriction fragment length polymorphism (PRA) based on hsp65 gene is preferred since it offers an easy، rapid، and inexpensive means of identifying pathogenic mycobacterial isolates to species level. A combination of phenotypic tests and PCR-restriction fragment length polymorphism (PRA) method targeting 441 bp hsp65 DNA used to find species diversity of Iranian clinical strains of mycobacteria. The test strains consisted of 270 clinical isolates of mycobacteria recovered from 2358 patients in two reference laboratories. A total of 207 isolates belong to M. tuberculosis were initially identified using conventional phenotypic techniques and specific PCR، based on detection of IS 6110. The isolates belonging to non tuberculosis mycobacteria (NTM) were subjected to further definitive identification using batteries of phenotypic tests and hsp65-PRA. Out of 270 clinical strains، 207 isolates were found to be M. tuberculosis by phenotypic techniques and specific PCR based on detection of IS 6110. NTM strains (63 isolates) represented a variety of the species comprised of 12 M. simiae، 9 M. fortuitum، 5 M. gordonae، 5 M. abscessus، 5 M. kansasii and some rare species including 3 M. massilainase، 3 M. thermoresitbile، 2 M. senegalense type 2، 1 M. conceptionense type 1 or M. senegalense type 1، 1 M. phlei، 1 M. chelonae، 1 M. nonchromogenicum، 1 M. genavense، 1 M. montefiorense or M. triplex، 1 M. branderi، 1 M. novocastrense، 1 M. nebraskense، 1 M. lentiflavum and 1 M. avium. This study showed that hsp65-PRA technique offers a simple، rapid، and accurate method for the identification of NTM clinical isolates.

Aim and The two genera Chenopodium and Atriplex contain 100 and 150 species، respectively; both of which belong to Chenopodiaceae family. Due to morphological similarity، the recognition of species of these two genera is comparatively difficult، thus، the anatomical characters are considered in the southern Khorasan Province. At first، for studying anatomical structure of species، herbal samples were gathered from different areas of this province. Then، the method of manual cutting was used in laboratory، and for staining tissues and better distinguishing their organizing sections، the method of with green methyl and Carmen stain. After preparing slides، photo was provided for all sections (leaf، stem، petiole and epiderm) and anatomical characters were evaluated by Olympus microscope equipped with ruler. The results show the characters of leaf and stem were distinctive in genus، but they couldn’t distinguish the species of each genus. In the species of C. botrys glandular trichomes، to be thick of its coticule in both surfaces، the form of petiole section and type of stomata is below surface in both level of epiderm، to distinguish it from other species of Chenopodium genus. The most differences have been seen in the petiole character signifying the recognition of the species.

Aim and Exopolysaccharides are natural polymers that are produced by Lactic acid bacteria (LAB)، Bifidobacteria and Bacillus. Due to their characteristic physical and chemical properties، EPS are widely used in food industry as viscosifying، stabilizing، gelling، or emulsifying agents. EPSs synthesized by Bacillus spp. have comparatively elevated viscosity and superior pseudoplastic properties; there has been increasing interest about them in recent years. The aim of the present study was isolation of two Exopolysaccharides (the cell-bound EPS (EPS-b) and the released EPS (EPS-r)) produced by10 strains of Bacillus spp. isolated from agricultures and identification of a strain that produces the highest amount of EPS. 10 strains of Bacillus were overnight pre grown in MRS broth medium containing 2% (w/v) glucose at 37°C for 24-48h under aerobic conditions. The total amount of carbohydrate in the EPS was determined using the phenol/sulfuric acid method with glucose as the standard. Among 10 Bacillus spp، two strains had the highest amount bound EPS (EPS-b)، maximum amount reached 43 mg/l (B5) and minimum amount reached 0. 2 mg/l (B1، B4، B8، B9، B100). All strains synthesized EPS-r that maximum amount reached 229 mg/l (B7) and minimum amount reached 45mg/l (B8). Growth kinetics of the chosen strain (B7) in liquid medium with the production of EPS was investigated. The results show that the maximum production of EPS of the chosen strain (B7) was obtained in exponential growth phase. The level of EPS was then quite stable. Results of this study showed that Iranian potential probiotic Bacillus species. have potential of EPS production.

Aim and Brucella spp. is a gram negative coco bacillus which has different hosts including human، cow، sheep and goat. Brucella spp. is transferred via milk and dairy products causing a Malta fever in human. Serological methods for Brucella spp. detection do not have sufficient sensitivity and accuracy; in addition، they have great false positive and negative results. The aim of this study is determination of the hemi nested PCR technique sensitivity for detection of Brucella spp. in raw milk and cheese. In this study 15 raw milk samples، 15 pasteurized cheese samples and 15 traditional cheese samples were collected from Tehran and suburbs. DNA was extracted using DNP kit from samples. After optimization of first and second round of PCR، sensitivity and specificity of reactions were examined. Then all samples were tested using developed PCR. The PCR product which was obtained from first PCR round had 443 bp length، and the second round fragment had 225bp. The PCR sensitivity up to 100 particles was observed. Among 15 samples of raw milk، 10 cases were PCR positive. In 15 pasteurized cheese samples only 6 cases were PCR positive، and finally from 15 traditional cheese samples 8 were PCR positive. With respect to this result، it could be said that in comparison with conventional detection technique for Brucella spp. diagnosis، PCR has a significant sensitivity and accuracy. Furthermore، it seems that using molecular detection technique as confirmative tests for detection of Brucella spp. is inevitable.

Aim and The main purpose of this research is cisplatin loading on liposome for targeted drug delivery. The suitable loading percentage obtained showed that we have achieved the target. Liposome was extracted from a lecithin source and after dissolving in phosphate buffer the solution was sonicated in bath sonicator for loading the cisplatin on extracted liposomes and resizing to nano scale. Total amount of plating in sonicated solution is known. Platin which exists in solution transparent phase after ultra centrifuge process، was determined with atomic absorption test. By subtracting these two amounts from each other we achieved the amount of platin existed in sedimentation phase. Particles’ size in nano scale were confirmed with zeta sizer. Liposome is known as a new lipid carrier for targeted drug delivery in cancer therapy. The anti cancer drug “cisplatin”، was encapsulated in liposome to reduce the side effects of drug on other cells and have a more dosage of drug on target cell.

Aim and Environmental protection requires taking into account the genotoxic hazards likely to concern different populations of ecosystems. Genotoxins are substances that cause heritable changes in the genetic material in germ cells، namely spermatocytes or oocytes. Since such hazards may emerge within waters، it is essential to assess them by means of laboratory tests that allow the evaluation، within aqueous environments of the genotoxicity of a water effluent substance or preparation with respect to organisms living in aquatic environments. This article describes a method that is likely to provide information in this field. Within aqueous environments it highlights، the genotoxic effects on larvaes of the amphibian species، Xenopus laevis. Xenopus laevis is a species of South African aquatic frog of the genus Xenopus. African clawed frogs can grow up to a length of 12 cm. They have a flattened head and body، but no tongue or external ears. The tested organisms were exposed to a range of concentrations for 12 days. The negative and positive controls were carried out in parallel under the same conditions. The positive control allows the quality of the biological reagent to be checked and the test to be validated. The rate of erythrocytes with micronuclei was determined for each test concentration and control solutions. The rate of erythrocytes with micronuclei for each concentration was compared to that of the negative control in order to determine the concentrations that induce a positive genotoxic effect. The positivity of a test can be based on the detection، with respect to the negative control، of a statistically significant response for at least one of the test concentrations and the maximum concentration which does not give rise to an acute toxicity has been determined and tested.