4 mL of 99% ethanol. Two hundred microliter samples were then read on a Spectra Max Plus Spectrophotometer at 560 nm and concentrations determined by comparison with cysteine standards. Enzymatic activities are presented on a MK-2206 cell line per protein basis. Cysteine desulfhydrase activity was determined by following a modified protocol from Chu and colleagues . One hundred microliter samples in 10mM potassium phosphate buffer were transferred to 1.5 mL microcentrifuge tubes. The reactions were initiated by the addition of 900 μL 0.11 mM L-cysteine followed by vortexing and incubated at 37°C for 1 h. Sulfide production was quantified by following the protocol described above in the sulfide
analysis section . Protein assays Bradford assays were determined by following the protein microplate bioassay procedure supplied by Bio-Rad (Mississauga, Canada). AZD6738 mw Protein Assay Dye Reagent concentrate was diluted 5 times in distilled water. Ice-cold samples were homogenized using a Bullet Blender (Next Advance, Averill Park, NY) for 5 minutes on its maximum speed. The homogenized cells were then transferred into fresh 1.5 mL microcentrifuge tubes and centrifuged at 1000 g for
5 min to pellet cellular debris. Then 80 μL samples from the supernatant were diluted with 720 μL of double deionized water. To this 200 μL of dye reagent was added to each tube, vortexed and the samples incubated at room temperature for 5 minutes. Two hundred microliter aliquots were then read at 595 nm in a Spectra Max Plus Spectrophotometer. Statistics Liothyronine Sodium Analysis of variance (ANOVAS) and Tukey-Kramer post hoc tests were performed using JMP 8.0 software (SAS Incorporated.), or where appropriate, T-tests
were analyzed using Microsoft Excel 2007. All experiments include representative standard errors (SE). Experiments were performed at least in triplicate and the results are indicative of n = 3 for enzymatic assays. SE is presented in all figures by the error bars. Where it is not visible, SE is smaller than the character at that point. Acknowledgements This research was supported by Natural Sciences and Engineering Council of Canada and the Advisory Research Committee of Queen’s University. References 1. Elinder CG, Kjellström T, Hogstedt C, Andersson K, Spång G: Cancer mortality of cadmium workers. Br J Ind Med 1985, 42:651–656.PubMed 2. Garcia-Morales P, Saceda M, Kenney N, Kim N, Salomon D, Gottardis M, Solomon H, Sholler P, Jordan V, Martin M: Effect of cadmium on estrogen receptor levels and estrogen-induced responses in human breast cancer cells. J Biol Chem 1994, 269:16896–16901.PubMed 3. Sataruga S, Haswell-Elkinsa MR, Moorea MR: Safe levels of cadmium intake to prevent renal toxicity in human subjects. Br J Nutr 2000, 84:791–802. 4. Heng L, Jusoh K, Ling C, Idris M: Toxicity of single and combinations of lead and cadmium to the cyanobacteria Anabaena flos-aquae . Bull Environ Contam Toxicol 2004, 72:373–379.PubMedCrossRef 5.