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Heravi R.M.,Ferdowsi University of Mashhad | Nasiraii L.R.,Islamic Azad University | Sankian M.,Bu Ali Research Institute | Kermanshahi H.,Ferdowsi University of Mashhad | Varasteh A.R.,Mashhad University of Medical Sciences
Biotechnology | Year: 2012

To date, lactobacilli are widely used in food industries and new probiotic products; hence these are considered as an attractive target for genetic modifications. This study was conducted to improve electroporation of probiotic lactobacilli which is a necessary prerequisite for genetic manipulations. Four strains of probiotic lactobacilli from different sources were grown in Man Rogosa and Sharp (MRS) broth medium containing glycine, as a cell wall weakening agent or a pulse of glycine for evaluation of glycine effect on electroporation efficiency. After evaluation of various parameters such as washing buffers, washing times, electric field strength, pulse duration and plasmid concentration, a practical electroporation protocol was presented to improve electrotransformation of lactobacilli in comparison with two standard protocols. This new protocol exhibited higher transformation efficiency (transformants/μg plasmids) than standard methods (p<0.05) with no differences between species (Lb. casei, Lb. crispatus, Lb. salivarus and L. rhamnosus). The pulse of glycine had no effect on the number of the transformants in three methods. In contrast to plasmid concentration, plasmid size had no influence on the transformation efficiency. The modified method enabled to transform plasmid into the resistant lactobacilli against transformation. These bacteria have potential for bioengineering research to improve special performance. © 2012 Asian Network for Scientific Information.

Sankian M.,Bu Ali Research Institute | Talebi F.,Bu Ali Research Institute | Moghadam M.,Bu Ali Research Institute | Vahedi F.,Razi Vaccine and Serum Research Institute | And 3 more authors.
Allergology International | Year: 2011

Background: Oral allergy syndrome resulted from plant-derived foods is frequent among adults. Allergy to melon (cucumis melo) is one of the most frequent fruit allergies in Iran. Three different major allergens have been found in Cucumis melo that Cuc m 1 (cucumisin) has been identified as the major allergen of melon. Cucumisin is an alkaline serine protease that it is found as a 78 kDa protein in precursor form. The aim of this study was production of recombinant Cuc m 1 in Escherichia coli (E. coli) cells and characterization of its allergenicity property. Methods: Production of recombinant Cuc m 1 was carried out by cDNA cloning technique into the pET32b(+) vector using specific primers designed based on cucumisin nucleotide sequence available in Genebank database, cucumisin encoding gene and directional cloning method. Cloned plasmid into E. coli TOP10 was transformed into E. coli BL21 and expression of the protein was induced by IPTG. The recombinant protein was purified via Ni-NTA affinity chromatography using histidine tag in recombinant protein. IgE binding of this protein was assessed by IgE-immunoblotting, ELISA and inhibition ELISA. Results: The directional cloning was resulted in expression of a fusion Cuc m 1. Immunoblotting with sera of patients allergic to melon showed strong reactivity with purified protein band. Inhibition assays demonstrated that purified rCuc m 1 could be the same with natural form of Cuc m 1 in total extract. Conclusions: In the present study, we have provided a functional recombinant cucumisin allergen, rCuc m 1 with 86 kDa, which may be used as a standard allergen for clinical diagnosis and study of allergy to melon. © 2011 Japanese Society of Allergology.

Assarehzadegan M.A.,Ahvaz Jundis hapur University of Medical science | Assarehzadegan M.A.,Bu Ali Research Institute | Sankian M.,Ahvaz Jundis hapur University of Medical science | Sankian M.,Bu Ali Research Institute | And 6 more authors.
Allergology International | Year: 2010

Background: The inhalation of Salsola kali pollen is an important cause of pollinosis during summer and early fall throughout desert and semi-desert areas. Sal k 1 has been previously reported as a major allergen of S. kali pollen. In this study, we produced the recombinant Sal k 1 and also its low IgE-binding mutant form. We further compared the IgE binding ability of these two recombinant molecules. Methods: The recombinant Sal k 1 and its low IgE-binding variant, obtained by three amino acid exchanges (R142→S, P143→A, D144→V), were cloned and expressed in E. coli, as proteins fused with thioredoxin and His-tags, and then purified by Ni2+ affinity chromatography. The IgE-binding capacity of the wild-type and mutated rSal k 1 was compared using immunoblotting, ELISA and inhibition assays by ten sera from S. kali allergic patients. Moreover, in vivo IgE-reactivity was investigated by the skin prick test. Results: Both the recombinant and the mutated form of Sal k 1 were expressed in E. coli at a relatively high amount and soluble form. All sera recognized rSal k 1 via immunoassay analysis. In addition, inhibition assays demonstrated that the purified rSal k 1 was similar to its counterpart in the crude extract. The mutated rSal k 1 exhibited a reduced IgE-binding capacity against wild-type rSal k 1. Conclusions: This study demonstrates that purified rSal k 1 is comprised of IgE-epitopes similar to that of its natural counterpart and that the mutated variant showed a reduced IgE-binding capacity based on in vitro assays and in vivo provocation testing. © 2010 Japanese Society of Allergology.

Kohbanani M.S.,Mashhad University of Medical Sciences | Kohbanani M.S.,Rafsanjan University of Medical Sciences | Nikravesh M.R.,Mashhad University of Medical Sciences | Jalali M.,Mashhad University of Medical Sciences | And 3 more authors.
Pakistan Journal of Biological Sciences | Year: 2012

Maternal smoking has been clearly demonstrated to be associated with increased health problems in infants and children. Nicotine is the chemical substance with high level of toxicity. It crosses through the placenta and accumulates in the developing organs of fetus. Previous investigation indicated that maternal nicotine exposures induce decreased fibronectin expression in lung parenchyma. In this study, the effect of maternal nicotine exposure on laminin expression of the newborn mice lungs has been evaluated. Female pregnant Balb/C mice were divided randomly in to four groups as fallow: Experimental groupl (Exp D1); was received 3 mg kg-1 nicotine intra peritoneal injection (IP) from gestational day 7 (GD7) to the last day of pregnancy, Experimental group 2 (Exp D14); was received 3 mg kg-1 nicotine from GD7 to postnatal day 14, Groups 3 and 4; as sham control groups (Sha-Con) were received the same volume (3 mg kg-1) of normal saline parallel to experimental groups. At the end of exposure times, all of newborns were anesthetized; their lungs were removed and prepared for immunohistochemical method and real-time polymerase chain reaction. The finding indicated that laminin alpha 5 (Lama5) mRNA expressions in the lung of newborn in the nicotine treated Exp Dl decreased by 0.63 fold but increased in Exp D14 by 1.57 fold comparing to Sh-Con groups. Lama5 immunoreactivity was not similar in different parts of the lungs including alveoli and bronchiole, having a significant increase in the experimental groups in contrast to the Sh-Con groups. However, increase in immunoreactivity observed more in Exp Dl 4. Immunoreactivity intensity in small vessels of all experimental groups was not significantly different. These data also indicate that maternal nicotine exposure may induce abnormal laminin expression which may cause defects in lung function during life time. © 2012 Asian Network for Scientific Information.

Ajami B.,Harvard University | Ghazvini K.,Ghaem Hospital | Movahhed T.,Harvard University | Ariaee N.,Bu Ali Research Institute | Makarem S.,Mashhad University of Medical Sciences
Iranian Red Crescent Medical Journal | Year: 2012

Background: Dental unit waterline system is considered potential source for contamination with Legionella species. The aim of this study was to determine if contamination of a dental unit water line system by Legionella pneumophila serogroup1 in the Mashhad School of Dentistry occurred in 2009. Methods: A total of 52 dental units were selected from all clinical departments of the Mashhad School of Dentistry. Samples of water were collected from outlets of water/air spray, high-speed dental hand pieces and water cup fillers. Samples were tested via the ELISA method. Results: At the beginning of the work day, a total of 36.1 percent of dental units were contaminated by Le-gionella pneumophila serogroup 1. Conclusion: Infection control of the dental unit water line system regarding legionella in the Mashhad School of Dentistry is a challenge and engineering controls should be used in contaminated clinics. © Iranian Red Crescent Medical Journal.

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