[Optimization of gene expression and purification of Legionella pneumophila peptidoglycan associated lipoprotein recombinant protein]

There are many problems with most of the available diagnostic tests used to diagnose Legionella pneumonia, including inadequate sensitivity and specificity, and inability to provide a result in a clinically useful time period. Legionella pneumophila PAL protein has been considred as a target for detecting of Legionella infection from urine specimen, because it is conserved sequence and is secreted into the urine. The aim of this study was to optimize expression and purification of L. pneumophila PAL protein. In this experimental study, optimizing of 5 parameters [cell density, induction time, growth temperature, IPTG concentration and type of medium] was performed. After expression, periplasmic extract was prepared and recombinant PAL protein purified using Ni2+-charged resin column. Finally, recombinant PAL protein was verified by Western blotting. In terrific broth medium, the optimum condition of r-PAL protein induction was occurred at an OD600 of 0.6, 1mM IPTG concentration and 15 hours incubation at 25°C Recombinant periplasmic PAL protein was highly purified [>80%] using Ni-NTA column. Western blotting analysis showed that recombinant PAL protein was also specifically recognized by anti-His6-peroxidase antibody. By purification of recombinant PAL protein in purity greater than 80% it can be used to evaluate its capacity in diagnosis of Legionella infection and preparation of diagnostic kit

Effect of Anthum graveolens fruit extract on rat uterus contractions

Dill [Anethum graveolens] from Umbelliferae is used traditionally to treat convulsions and increasing milk production. Also, in Darab [Fars Province] and Yazd dill fruit is used traditionally to prevent abortion. Its antimicrobial, antihyperlipidaemic, anti-hypercholesteroaemic effects and reducing gastric acid secretion have been reported. The aim of the present study was to investigate the effects of dill fruit hydroalcoholic [DFHE] extract on virgin rat uterus contractions induced by KCI [60mM] and oxytocin [10 mU/ml]. Macerated method was used for extract preparation. Virgin Sprague Dawley female rats were pretreated with an injection of estradiol valerate [5mg/kg, s.c.] 24 hours prior to experiment. Rats were killed and uteri were dissected and mounted in an isolated organ bath containing De Jalon solutions [29°C] bubbled by oxygen. Contractile responses were recorded isometrically under 1g initial tension. The DFHE [0.5, 1, 2, 4 and 8 mg/ml] relaxed both KC1- and oxytocin-induced contractions dosedependently [p<0.0001]. However, the spasmolytic effect of extract on oxytocin-induced contractions was more potent. In Ca[2+] -free, rich K+ [120mM] De Jalon solution, cumulative adding of calcium [0.15, 0.3, 0.6, 1.2 and 2.4 mM], increased contractions dose dependently [p<0.0001]. DFHE [4 mg/ml] shifted this dose-response curve to the right [p<0.05]. In Ca[2+] free solution, oxytocin induced strong contractions if calcium [0.3 mM] was added to the bath. However, DFHE [8 mg/ml] reduced calcium-induced contraction [p<0.001]. The spasmolytic effect of DFHE was reversible. The spasmolytic effect of DFHE [4 mg/ml] was affected neither by propranolol [1micro M] nor by L-NAME [100 micro M]. These results suggest that the relaxatory effect of DFHE on KCI and oxytocin-induced uterus contractions is due, at least in part, to blockade voltage dependent calcium channels and partly by disturbing in releasing calcium from intracellular pool indirectly. In addition, the beta-adrenoceptors and NO are not involved in this inhibitory effect of DFHE. Our results may also support the traditionally usage of dill fruit for preventing abortion