Iranian Biomedical Journal
Year:2007 | Volume:11 | Issue:3|Papers Count:10

Background: Guaiacol peroxidases (GP) are haem-containing enzymes participating in many physiological processes in plants. The expression pattern of these enzymes is organ-specific and developmentally regulated. Methods: The presence of GP activity in extract samples, prepared from Crocus sativus L. corms that were either dormant or rooting for 3, 6 and 10 days, was investigated. Results: Kinetic studies revealed a significant similarity among GP activities detectable in the corm at different stages of development: in all extract samples, the activity was maximal at pH 7.5 and after preincubation at 30-40oC. When guaiacol was used as the varying substrate, Michaelis-Menten kinetics behavior was observed in all extract samples and resulted in similar KM values; catalytic efficiencies were also very similar. The corm GP activity was inhibited by cyanide, azide and ascorbate. The GP activities from different extract samples had the same sensitivities to azide, cyanide and ascorbate and the type of inhibition by azide and cyanide was competitive and uncompetitive, respectively, while ascorbate inhibited the GP activity non-competitively. Corm extract samples from different stages of rooting similarly responded to temperature treatment and a biphasic Arrhenius plot resulted for each extract sample studied. When dormant, 3-, 6- and 10-days-rooting corm extracts were submitted to non-denaturing polyacrylamide gel electrophoresis, the GP-specific activity staining revealed one band on the gel, with the same migrating distances. Conclusion: This finding in combination with kinetic studies demonstrated that at least one form of GP, with an apparent molecular weight of 68 kDa, was expressed during development of Crocus sativus L. corm.

Background: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. Methods: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. Results: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. Conclusion: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.

Background: Knowing that adenoviral vectors could initiate innate immunity, the ability of E1-deleted recombinant adenovirus (Ad-E1D) in induction of B7.1 and IL-2 molecules was studied. Methods: The expression of green fluorescent protein in C1498 cells following transfection of these cells with adenovirus green fluorescent protein vector confirmed the ability of adenovirus vectors in infecting the cells and inducing the expression of the gene of interest. The expression of B7.1 molecule on the surface of the cells was assayed upon infection with Ad-E1D vector. Adenovirus-IL-2/B7.1 vector capable of inducing IL-2 and B7.1 expression in the cells was used as the positive control vector. Results: According to the FACS results, about 4.17% of normal cells expressed B7.1 on their surface, while this level was increased in Ad-E1D transduced cells up to 14.43%. These results demonstrate that Ad-E1D vector considerably (about 3 folds) increases the expression of B7.1 on the cells. No detectable IL-2 was secreted into the medium of non-transduced and Ad-E1D transduced cells. Conclusion: Data indicate that the infection of C1498 cells with recombinant adenoviruses stimulates expression of B7.1 on the cell surface rather than secretion of IL-2 into the medium. Iran.

Background: Enterococci are important because of their role as the leading cause of nosocomial infections which have a significant role in the dissemination and persistence of antimicrobial resistance genes. Methods: In this study, we determined the distribution of enterococcal species in the sewage treatment plants in Iran. Furthermore, we improved a rapid and specific PCR method using primers (sodA and ddl genes) for identification of enterococci spp. Results and Conclusion: A total number of 712 enterococci spp. Were isolated and the results showed that 56%, 24%, 12%, 4%, 2%, 1% and 1% isolates were E. faecium, E. hirae, E. faecalis, E. gallinarum, E. casseliflavus, E. mundtii and other enterococcal spp., respectively. The use of species-specific PCR was in agreement with the biochemical tests. Furthermore, multiplex PCR was developed to study the presence of vancomycin resistant genes in E. faecium or E. faecalis. The multiplex PCR appeared to be a useful, rapid and specific method for detecting and discriminating genotypes for vancomycin-resistant Enterococcus.

Background: Anopheles culicifacies is a main malaria vector in southeastern part of Iran, bordring Afghanistan and Pakistan. So far, resistance to DDT, dieldrin, malathion and partial tolerance to pyrethroids has been reported in An. stephensi, but nothing confirmed on resistance status of An. culicifacies in Iran. Methods: In current study, along with WHO routine susceptibility test with DDT (4%), dieldrin (0.4%), malathion (5%), permethrin (0.25%), lambadacyhalothrin (0.1%), and deltamethrin 0.025, we cloned and sequenced segment VI of domain II (SII6) in voltage-gated sodium channel (vgsc) gene of An. Culicifacies specimens collected in Sistan and Baluchistan province (Iran). Results: A 221-bp amplified fragment showed 91% and 93% similarity with exon I and exon II of An. gambiae. The size of intron II in An. culicifacies is 62 bp, while in An. gambiae is 57 bp. The major difference within An. culicifacies specimens and also with An. gambiae is in position 29 of exon I, which led to substitution of Leu to His amino acid. Conclusion: This data will act as first report on partial sequence of vgsc gene and its polymorphism in An. culicifacies. A Leu to His amino acid substitution detected upstream the formerly known knockdown resistance (kdr) mutation site could be an indication for other possible mutations related to insecticide resistance. However, the result of WHO susceptibility test carried out in Baluchistan of Iran revealed a level of tolerance to DDT and dieldrin, but almost completes susceptibility to pyrethroids in An. culicifacies. We postulate that the molecular diagnostic tool developed for detection and identification of kdr-related mutations in An. culicifacies, could be useful in monitoring insecticide resistance in Iran and neighbouring countries such as Pakistan and Afghanistan. A phylogenetic tree also constructed based on the sequence of exon I and II, which readily separated An. culicifacies populations from An. stephensi, An. fluviatilis and An. gambiae.

Background: It is well established that the esophageal distention (ED) leads to gastric relaxation, partly by vago-vagal reflex, but till now, the effect of ED on gastric acid secretion has not been investigated. The aim of this study was to investigate the effect of ED on basal and stimulated gastric acid secretion. Methods: Adult male Wistar rats (200-240 g) were deprived of food but not the water 24 h before the experiments. Under urethane anesthesia (1.2 g/kg, i.p.), animals underwent tracheostomy and laparotomy. A catheter was inserted in the stomach through duodenum for gastric distention and gastric washout and the esophagus was cannulated with a distensible balloon orally to distend esophagus (0.3 ml, 10 min). Gastric acid secretion was stimulated by gastric distention, carbachol (4 mg/kg, i.p.) or histamine (5 mg/kg, s.c.). Effects of vagotomy, NG–nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.) and also hexamethonium were investigated. Results: Basal and gastric distention- and carbachol, histamine-stimulated acid secretion were reduced by the ED (P<0.05, P<0.0001, P<0.01 and P<0.02, respectively). L-NAME (10 mg/kg, i.v.) elevated the acid output (P<0.002). Vagotomy reduced the inhibitory effect of the esophagus distention on gastric distention-induced acid secretion (P<0.01). Conclusion: These results indicate that the vagus nerves are involved in the inhibitory effect of the ED on the basal and stimulated gastric acid secretion. Furthermore, nitric oxide could be involved.

Introduction. The non-enzymatic glycation of Low density lipoprotein (LDL) is a naturally occurring chemical modification of apolipoprotein B as a result of condensation between lysine residues and glucose. Glycated LDL is poorly recognized by LDL receptors and initiates different processes that can be considered proatherogenic. Thus, LDL glycation may contribute in the increased atherosclerotic risk of patients with diabetes. The objective of this study was to investigate the effect of naturally occurring flavonols on LDL glycation in vitro. Methods: In this study, LDL was isolated from EDTA-plasma by ultracentrifugation using a single step discontinuous gradient. Then, glucose was added to LDL and LDL glycation level was estimated in absence and presence of flavonols by sodium periodate assay. Results: This study was showed that five flavonols: quercetin, myricetin, kaempferol, rutin and morin decreased LDL glycation in a dose-dependent manner. Also, it was demonstrated this nutrients decreased electrophoretic mobility of glycated LDL. Conclusion: The results of this investigation show that flavonols probably with their antioxidant properties inhibited LDL glycation and thus may have a role in ameliorating atherosclerotic risk of patients with diabetes mellitus.

Background: Accurate measurements of physical characteristics of bone are essential for diagnosis, assessment of change following treatment, and therefore, indirectly, for evaluation of new forms of therapy. This is particularly true of osteoporosis and aging skeleton, in which fractures occur easily. Methods: In this study an ultrasonic system was set-up and calibrated on Plexiglas tubes of variable thickness then used to detect the cortical bone thickness change in calf and bovine adult femurs. Lamb waves have been generated and detected using a pair of Piezoelectric point transducers (transmitter and receiver) operating at 60 kHz in contact with the surface of the bone. Results: A link has been established between the ultrasound velocity and the bone thickness. On the other hand, the density variation has been also investigated by the simulation of the bone decalcification chemically. The results show that the velocity is very sensitive to both thickness and density, its value reduces as the cortical bone thickness and density decrease. Conclusion: This technique might be considered in the attendance of certain bone diseases expressing itself by gradual change in physical properties.

Background: Adenoviruses are used extensively to deliver genes into mammalian cells, particularly where there is a requirement for high-level expression of transgene products in cultured cells, or for use as recombinant viral vaccines or in gene therapy. In spite of their usefulness, the construction of adenoviral vectors (AdV) is a cumbersome and lengthy process that is not readily amenable to the generation of large collection of clones. Methods: In this project, to delete E1 gene in adenovirus, an adenoviral plasmid containing lateral sites of E1 region of adenovirus was made and recombination in the 293A cells between the homologous region of this linearized plasmid and the adenovirus genome resulted in the formation of the complete adenoviral recombinant. Results: This recombination resulted in loss of E1 region and we constructed a recombinant adenovirus type 5 vector that E1 gene was deleted by homologous recombination. Conclusion: Homologous recombination is more easy and fast technique in the production of AdV.

Background: Colorectal cancer (CRC) is one of the most common forms of cancers in the world and is curable if diagnosed at the early stage. Analysis of DNA extracted from stool specimens is a recent advantage to cancer diagnostics. Many protocols have been recommended for DNA extraction from stool, and almost all of them are difficult and time consuming, dealing with high amount of toxic materials like phenol. Their results vary due to sample collection method and further purification treatment. In this study, an easy and rapid method was optimized for isolating the human DNA with reduced PCR inhibitors present in stool.Methods: Fecal samples were collected from 10 colonoscopy-negative adult volunteers and 10 patients with CRC. Stool (1 g) was extracted using phenol/chloroform based protocol. The amplification of P53 exon 9 was examined to evaluate the extraction efficiency for human genomic targets and also compared its efficiency with Machiels et al. and Ito et al. protocols. Results: The amplification of exon 9 of P53 from isolated fecal DNA was possible in most cases in 35 rounds of PCR using no additional purification procedure for elimination of the remaining inhibitors. Conclusion: A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols.