PubMed · 6581319
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J D Bogden. 1983. Trace element nutrition.. https://pubmed.ncbi.nlm.nih.gov/6581319/
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Lysine and leucine auxotrophic, heterothallic (h+, h-) strains of Schizosaccharomyces pombe were used to obtain chromium (VI)-sensitive and -tolerant mutants by ultraviolet radiation-induced and nitrosoguanidine-induced mutagenesis. The minimal inhibitory concentrations of K2Cr2O7 on YEA media were 225 microM for the wild-type strain CW-6, 125 microM for the sensitive mutant CS-6.51 and 275 microM for the tolerant mutant CT-6.66. The mutants exhibited cross-sensitivity of various patterns to Cd2+, Cu2+, Ni2+, Zn2+ and VO3-(4). Cr(VI) was added to the actively growing cultures and the total chromium (TOCr) content of the cells was determined. The sensitive mutant exhibited a high bioaccumulation ability, with a dry biomass of 810 micrograms g-1 after 30 min, while the tolerant mutant had a significantly lower ability than the wild-type strain. In PIPES buffer, washed, lysine-starved biomasses were treated with 75 microM Cr(VI) and after 2 h, the TOCr and the organically bound chromium (OBCr) were determined. Under these conditions, the sensitive and tolerant mutants had the same TOCr content, 50% of which was OBCr. The wild-type strain exhibited a lower TOCr content than that of its mutants and only 35% of this was OBCr. The Cr(VI)-sensitivity was due to a significantly increased uptake of Cr(VI).
A modified concentric nebulizer was used as the interface to couple capillary electrophoresis (CE) to inductively coupled plasma atomic emission spectrometry (ICP-AES). The CE capillary replaces the central tube of the concentric nebulizer. The tip of the nebulizer tapers slowly to allow uncertainty in the position of the capillary. A platinum wire was inserted into the CE capillary to provide electrical connection to the CE power supply. pH changes inside the capillary due to electrolysis of the background buffer electrolyte was small and has minimal effects on the CE separation. The peak broadening effects due to the nebulizing gas flow, however, were significant. Resolution decreases quickly when the flow-rate of the carrier gas increases. Sample stacking technique was used to improve the resolution of species of opposite charge, e.g., Cr(VI) vs. Cr(III) ions. Detection limit of Cr based on peak area is approximately 10 ppb for the CE-ICP-AES system.
A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.