ABSTRACT
<p><b>OBJECTIVE</b>This study aimed to investigate the susceptibility of mice with streptozotocin(STZ)-induced diabetes mellitus (TIDM) to the uptake of pentavalent inorganic arsenic (iAsV) and the possible molecular mechanism.</p><p><b>METHODS</b>TIDM was induced in mice by STZ. TIDM and normal mice were treated with 15.0 mg/kg Na2HAsO4·12H2O by intragastric administration. Then, the concentrations of arsenic in various tissues were measured by atomic fluorescence spectrometry. The gene expression levels of Pit1 and Pit2 were quantified by real-time RT-PCR, and their protein levels were detected by Western blotting in mouse heart, kidney, and liver tissues.</p><p><b>RESULTS</b>The concentrations of arsenic in STZ-induced TIDM mouse tissues were higher at 2 h after intragastric administration of Na2HAsO4·12H2O. Compared with the levels in normal mice, PIT1 and PIT2, which play a role in the uptake of iAsV, were upregulated in the livers and hearts of TIDM mice. PIT1 but not PIT2 was higher in TIDM mouse kidneys. The upregulation of Pit1 and Pit2 expression could be reversed by insulin treatment.</p><p><b>CONCLUSION</b>The increased uptake of iAsV in TIDM mouse tissues may be associated with increased PIT1 and/or PIT2 expression.</p>
Subject(s)
Animals , Male , Mice , Arsenic , Pharmacokinetics , Diabetes Mellitus, Experimental , Metabolism , Environmental Pollutants , Pharmacokinetics , Gene Expression Regulation , Physiology , Mice, Inbred ICR , Sodium-Phosphate Cotransporter Proteins, Type III , Genetics , Metabolism , Transcription Factor Pit-1 , Genetics , MetabolismABSTRACT
This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.