Comparing invasive and non-invasive of isolated Shigella flexneri by electron microscopy of cell culture, SDS-PAGE and Congo red method

BACKGROUND: The aim of this study was to compare invasive and non-invasive strains of Shigella flexneri isolated from Tehran by a 120 kDa protein band by SDS-PAGE, electron microscopy of cell culture and Congo red dye methods. METHODS: S. flexneri strains were isolated by standard bacterial methods from fecal specimens of children attending to the 3 children's hospitals. Phenotype analysis for screening virulent of strains of S. flexneri was done on a plate of tryptic soy agar contained 0.003% Congo red dye. Whole membrane protein preparations were used to examine the protein profiles of the inner and outer membrane of these Gram-negative bacteria. The protein mixture was electrophoresed through a polyacrylamide gel. The gel was stained with Coomassie brilliant blue R250 and destained with ethanol and acetic acid. HeLa cell culture was done by two-step preparations: one for light microscopy and the other for electron microscopy. RESULTS: Some of S. flexneri (46%) were Congo red positive colonies. S. flexneri with negative Congo red phenotype could not enter the HeLa cell culture. A 120 kDa protein band was found in 46% of these bacteria which could enter into HeLa cell culture. Pseudopod structures which facilitate bacterial cell-to-cell spread were readily identified by electron microscopy. DISCUSSION: Since the existence of 120-kDa protein band was corresponded to enter of S. flexneri into the HeLa cell culture and correlated with Congo red dye positive, for identification of invasive and non-invasive S. flexneri strains, the use of a 120-kDa protein band by SDS-PAGE or a simple, rapid and very cheap Congo red dye method is recommended. Because, there are some deaths due to Shigella sp. in our country, notification on the isolation of these bacteria in both children hospitals laboratories and private clinical laboratories is important.     

Growth performance,haematology, antioxidant status,immune response and histology of common carp (Cyprinus carpio L.) fed biofloc grown on different carbon sources

A 10 weeks trial was performed to investigate how different carbon sources (sugar beet molasses: SBM+BFT, sugar: S+BFT, corn starch: CS+BFT) along with control affect welfare status of common carp (Cyprinus carpio L.) fingerlings in biofloc‐based tanks. Three hundred healthy fingerlings (22.5 ± 0.2 g) were randomly distributed in 12 tanks (70 L) at a density of 8.02 kg/m³ (25 fish/tank). The fish in BFT treatments fed only 75% feeding rate of control. At the end of the experiment no differences were seen between the groups in case of growth performance, but the fish reared in CS+BFT had a significant lower food conversion ratio compared with the others (p < .05). Different carbon sources did not affect on haematological parameters (p > .05). Total serum protein and antibody concentration differed in treatments, and the highest values were found in S+BFT and CS+BFT treatments (p < .05). No significant differences were observed in case of lysozyme, superoxide dismutase and complement activity in treatments (p > .05), whereas the fish in BFT treatments showed a significant higher total antioxidant capacity and lower glutathione peroxidase than the control (p < .05). Different carbon sources resulted in no change in goblet and kupffer cells in intestine and liver respectively. The highest relative percentage survival was obtained in the CS+BFT and S+BFT (50%) in comparison with SBM+BFT (20%) treatment. The results obtained in this experiment, suggest that corn starch improves immune response, diseases resistance and histology of digestive and respiratory systems in carp fingerlings when used as a carbon source in zero water exchange system.     

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully. Key Words: Pteridine reductase 1 (PTR1), Leishmania, Gene expression     

In vitro antifungal activities of Allium cepa, Allium sativum and ketoconazole against some pathogenic yeasts and dermatophytes

By using an agar dilution assay, the antifungal activity of aqueous extracts prepared from Allium cepa (onion; AOE) and Allium sativum (garlic; AGE) were evaluated against Malassezia furfur (25 strains), Candida albicans (18 strains), other Candida sp. (12 strains) as well as 35 strains of various dermatophyte species and compared with the activity of a known antifungal drug, ketoconazole (KTZ). All the AOE, AGE and KTZ were found to be able to inhibit growth of all fungi tested in a dose-dependent manner with maximum of 100% at defined concentrations. The results indicate that onion and garlic might be promising in treatment of fungal-associated diseases from important pathogenic genera Candida, Malassezia and the dermatophytes.     

Allium vavilovii M. Popov et Vved. and a new Iranian species are the closest among the known relatives of the common onion A. cepa L. (Alliaceae)

A wild onion species from Karaj valley east of Tehran istaxonomically described as Allium asarense R. M.Fritsch et Matin. Sharing the bubble-like inflated lower scapewith A. vavilovii, the newspecies differs by semi-cylindrical leaves and small flowerswith translucent greenish-yellow tepals from all other knownspecies of Allium sect.Cepa. Molecular data suggest a closerelationship to A. cepa(but to a lesser degree than A.vavilovii) and indicate a still weakerphylogenetic connection of A.oschaninii to the common onion. The questionabout the possible wild ancestor of A.cepa is discussed against this background, and akey for determination of the Oschaninii-alliance of sect.Cepa is presented.