Construction and stable expression of a truncated human receptor tyrosine kinase ror1 [Ror1-ECD]

Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary results indicate the presence of a truncated transcript of Ror1 in tumor cells. The truncated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain [Ror1-ECD]. As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we construct-ed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line [CHO] was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules ex-pressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies

Expression of ROR1 in patients with renal cancer-a potential diagnostic marker

The ectopic expression of receptor tyrosine kinase Rorl has been reported in patients with hematological malignancies such as chronic lymphocytic leukemia and acute lymphoblastic leukemia. Here we report, for the first time, expression of ROR1 gene in both tumor tissues and peripheral blood mononuclear cells [PBMC] from patients with renal cancer [RC]. In the current study, the expression of ROR1 gene was semi-quantitatively measured in PBMC and tumor tissues from 16 RC patients as well as PBMC from 22 healthy individuals relative to the expression of the housekeeping gene phosphoglucomutase 1 by RT-PCR. Our results showed that ROR1 was expressed at gene level in 81.3% of renal tumor tissues [13 out of 16] whereas it was expressed in 94% of PBMC from RC patients [15 out of 16]. A weak expression of RORl was observed in PBMC of 4 out of 22 healthy individual. A significant expression of ROR1 was observed in PBMC from RC patients when compared to that in PBMC from normal healthy individuals [P<0.001]. The expression of ROR1 in PBMC may reflect a shedding of tumor cells into blood stream. We conclude that detection of a high level of ROR1 expression in blood cells might assist in early detection of renal malignancies, providing taking into consideration the clinical symptoms of the disease

Conjugation of monoclonal antibodies to super paramagnetic iron oxide nanoparticles for detection of her2/neu antigen on breast cancer cell lines

Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide [SPIO] nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20microg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques

Monitoring pyrethroid insecticide resistance in major malaria vector anopheles culicifacies: comparison of molecular tools and conventional susceptibility test

Anopheles culicifacies is a main malaria vector in southeastern part of Iran, bordering 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. 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]. 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. 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 complete 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