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

[Soluble CD26 and CD30 concentration in cutaneous leishmaniasis]

Introduction: Cutaneous leishmaniasis [CL] is the most common form of leishmaniasis that usually heals spontaneously with unsightly scar but rarely non-healing lesion of CL develops which is refractory to all types of therapy. It is shown that Th1 and Th2 response is associated with healing and non-healing form of the diseases, respectively. On the other hand, it is reported that CD26 and CD30 are associated with Th1 and Th2 types of response, respectively. In some diseases, there is a relationship found between level of CD26 and CD30 and Th1 and Th2 responses

Objective: The goal of this study was to determine the concentration of soluble CD26 and soluble CD30 [sCD26 and sCD30] as a possible marker for Th1/Th2 response in CL

Materials and Methods: The blood samples were taken from 36 patients with healing form of the lesion and 10 patients with non-healing form of CL who were referred to Razi hospital or Center for Research and Training in Skin Diseases and Leprosy in Tehran during 2003-2004. As a control blood samples were taken from 23 volunteers with no history of CL. In this study, the concentration of sCD26 and sCD30 were measured in plasma by ELISA method

Results: The results showed that the plasma levels of sCD26 and sCD30 were significantly higher in non-healing form of the disease than healing form of CL or control group [p<0.05]. The level of sCD30 was more prominent than sCD26. There was no significant difference in the level of sCD26 or sCD30 markers in healing form of CL compared to normal control group

Conclusion: Overall it seems that the level of sCD30 might be a useful marker to study the immune response in CL, which needs to be studied further