Cloning, expression and characterization of zebrafish ferroportin in Hek293T cell line

Ferroportin [Fpn], a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-Nl. The final resulted plasmid [pEGFP-ZFpn] was used for expression of Fpn-EGFP protein in Hek 293T cells. The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server [hidden Markov model], NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids. The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies

[ case - control study of prevalence of consanguineous marriage among parents of handicapped and healthy children in Ahvaz]

Previous studies showed that consanguineous marriage increases the incidence of mortality, congenital malformation and mental retardation in off springs. One of the most important factors in appearance of disability and genetic diseases is consanguineous marriage in Iran. This study aimed to comparing the prevalence of family marriage in disable and healthy children's parents in Ahvaz city, Iran. one hundred and thirty four disabled and 134 healthy children who were selected randomly in Ahvaz were evaluated in this case-control study and were matched by age and sex. Variables included consanguinity in parents, presence of a disabled person in the family, Mothers illness record and some parent's demographic factors. Data were collected by a questionnaire and analyzed by chi-square test, logistic regression and independent t-test. The study shows that the rate of family marriage in case and control groups was 79.1% and 57.4% respectively. There was a significant relationship between disability and consanguineous marriage, pregnancy age, history of convulsion and cyanosis, maternal diseases in pregnancy, being of a disabled family member and birth spacing from previous child [P<0.05]. There was, no significant relationship between ethnicity, mode of delivery, history of jaundice, and birth spacing from next child and disability [P>0.05]. Based on these findings consanguinity is the most important factor of genetic disorders and disability among the subjects studied. Therefore appropriate actions should be considered for decreasing the incidence of disability including: promotion of youth and families knowledge relating to risks of consanguineous marriage, available premarital genetic consulting and provision for prenatal screening tests

[ prevalence of candida species in saliva of asthmatic patients treated with inhaled corticosteroids: comparison of beclomethasone and fluticasone]

Candida is an opportunistic organism which will become pathogen if predisposing factors are present. One of the predisposing factors for increasing prevalence of candida is the use of corticosteroids. Researches on candida infections in treated patients with inhaled corticosteroids are controversial. The purpose of this study was evaluating the effect of two inhaler corticosteroids [Fluticasone and Beclomethasone] on the number and frequency of candida species in saliva and its comparison with normal subjects. This experimental research was performed on three groups of individuals. The first group was 30 asthmatic patients used inhaler Fluticasone, the second group with the same number of patients used inhaled Beclomethasone and the third group was composed of 60 normal individuals as control. Subjects were selected after evaluating the medical history and assurance of absence of the predisposing factors. Subjects' saliva were gathered in sterilized tubes and cultured in chromagar media for evaluating the type of candida. The results were analyzed with oneway and tukey tests. Based on this study, the number of candida species was significantly different among the three groups. The number of candida species in patients used fluticusone was higher in comparison with the other two groups [p<0.001]. This study showed that the number of candida species in patients treated with corticosteroids inhaler was high in comparison with the control group. Thus for avoiding candida infection, proper oral health and prevention of predisposing factors in these patients are recommended

[Serological survey of anti-Toxoplasma gondii antibodies in goats, sheep and cattle in slaughter houses of Mazandaran province, Iran]

Toxoplasmosis is a widespread zoonosis caused by Toxoplasma gondii. In addition to economic losses, this disease is transfer able to human and regarding the special ecological conditions of this parasite in nature of Mazandaran province, it is necessary to determine the situation of the disease. The purpose of this study was to determine the frequency of antibodies against T. gondii in cattle, sheep and goats slaughtered in official slaughterhouses in Mazandaran province, in 2004. To determine the prevalence of toxoplasmosis in slaughtered animals, in a descriptive cross-sectional study, 639 blood samples were obtained from cattle, sheep and goats between December 2004 and April 2005, from the three main geographical zones of Mazandaran province, Iran. Nine slaughterhouses were randomly selected in Western, Central and Eastern regions. Sera were extracted from 5mL venous blood samples, by centrifugation at 2000xg for 10 min, and were stored at -20°C prior to testing. Sera were screened for T. gondii antibodies by immunofluorescence antibody test [IFAT] and serum samples showing a titre of 1:16 were further diluted to determine the end point. Data were analyzed using Chi-square [X[2]] test. Of the 639 serum samples examined, 27.5% [176/639] showed positive titers equal or higher than 1:16 by IFAT. The highest frequency of antibody titres [1:16 titre] were found in sheep and the least in cattle [9%]. The highest infection rates in cattle were seen in western region with 5.5% antibody titres of 1:16. In three regions, 35% of sheep sera showed positive titers with high frequency of 1:16 antibody titre [17.3%] in Western region. Thirty percent of goat sera in 3 regions were seropositive and the highest infection rate was seen in 1:16 titre [18.5%] in Western region. The results of this study show that the presence of T. gondii specific antibodies for sheep and goats in Northern Iran is high, and the consequent risk of acquiring toxoplasmosis from human consumption of sheep and goat meat may be greater in this region

[Evaluation of cellular immunity of patients with brucellosis by measuring interferon gamma [IFN-gamma] and interleukin-13 [IL-13] by flow cytometry and Elisa]

Introduction: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. The animal findings Show that brucella infection induces cell-mediated responses. Some cytokines have an Important role in resistance to brucella infection

Objective: This Study is don't to Poesent a orecise an useful unseparated whole blood of healthy normal and patients with acute and chronic brucellosis as the source of cells evaluation of interferon gamma [IFN-gamma] and interleukin-13 expression

Materials and Methods: Diluted whole blood samples of 27 patients with acute [14] and chronic brucellosis [n13], and sex and age-matched healthy volunteers [n=22] With Meanage of 35-33+21 were cultured in the presence of either mitogen; heat inactivated bacteria or medium alone. Intracellular IL-13 and IFN-gamma were measured by specific sandwich ELISA and flow Cytometry was detected by the number of cytokine-producing CD3+ cells

Results: Findings indicated that extrandintracellular specific IFN-Y in creased Cansiderably [P<0.001] in cute brucelles patients. not only IFN-gamma production but also the number of IFN-gamma- producing CD3 cells were significantly decreased in response to antigen in chronic group of patients. There was a reverse correlation between the number of IFN-gamma-producing and IL-13-producing CD3 cells only in acute group which shows polarization of immune responses to Th1 in them

Conclusion: Although the percentage of CD3 IL-13-producing cells was dramatically high in the chronic group of patients, no correlation was found between the number of IFN-g-producing and IL-13-producing CD3 cells. In conclusion, the correlation of Th2 cytokines production and progression of chronic human brucellosis was not demonstrated. Nevertheless, diminished production of Th1 cytokines production in chronic group may suggest T cells unresponsiveness to Brucella antigen which helps prolongation of brucellosis in chronic patients