In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

Trace elements in rock salt and their bioavailability estimated from solubility in acid

In this study, 18 partly commercially available samples of rock salt from Austria, Germany, Pakistan, Poland, Switzerland, and Ukraine were investigated with respect to their content of trace elements using instrumental neutron activation analysis. Elements detected were Al, Ba, Br, Ca, Ce, Cl, Co, Cr, Cs, Eu, Fe, Hf, La, Mn, Na, Rb, Sb, Sc, Sm, Sr, Ta, Tb, Th, and Zn, some of them only in individual cases. An estimation of the bioavailability of these trace elements was performed by dissolving an equivalent of the sodium chloride samples in diluted hydrochloric acid (simulating stomach acid), filtering off the insoluble components, and analyzing the evaporated filtrate. It could be shown that in most cases bioactive trace elements like Fe can be found in rock salt in the form of almost insoluble compounds and are therefore not significantly bioavailable, whereas thorium, for example, was partly bioavailable in two cases. A significant contribution to the recommended daily intake of metal trace elements by using rock salt for nutrition can be excluded.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B.

Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.     

A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase

Background  

Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Entropy-driven polymerization of protein from the E66 strain of tobacco mosaic virus

Protein of the tobacco mosaic virus mutant E66 has lysine replacing asparagine of the type strain, vulgare, at position 140. Thus, E66 protein should have one more positive or one less net negative charge than vulgare at pH 6 to 7. To investigate the effect of charge, a comparative study of the polymerization of E66 and vulgare proteins at pH 6.0, 6.2, 6.4, 6.6, and 6.8 at ionic strengths 0.15, 0.10, and 0.05 was made by turbidimetry. Polymerization of E66 protein always proceeded at a lower temperature than vulgare. However, the extent of polymerization was much lower in E66, especially at the higher ionic strengths. Sedimentation velocity results paralleled those from turbidity measurements in that E66 protein polymerizes at lower temperatures than vulgare; the 20 S component is more abundant in E66 protein. Osmotic pressure measurements also show that E66 protein is more polymerized than vulgare, especially at lower pH values. Hydrogen ion titrations of E66 protein were carried out from pH 8 to 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. These titrations were reversible when carried out slowly. The isoionic point is near pH 5; thus the charge at pH 7.5 is ?3. The reversible titration results were correlated with the aggregates present at the various pH values and temperatures, determined from the areas under the schlieren peaks in sedimentation velocity experiments. It is found that hydrogen ion binding at the three pH values is correlated with the disappearance of the smallest aggregates and is independent of the type of higher polymer formed. To investigate the effect of ionic strength and pH on the characteristic temperature corresponding to an optical density increment of 0.01 by the method used previously for vulgare, two sets of turbidity measurements were carried out. In the first one the ionic strength was changed from 0.025 to 0.15 in increments of 0.025 at pH 6.0 and 6.4. In the other set, the ionic strength was kept constant at 0.10 and the pH changed from 5.9 to 6.7 in increments of 0.1 pH units. When the analysis of these data was carried out, $\text{Δ}\text{H}{}^1\text{= 30}\text{kcal/mol}$ was obtained. For the salting out constant a value of 1.7 was found, compared to 2.2 for vulgare, a result consistent with the fact that E66 should be less hydrophobic than vulgare. The electrical work term ΔW_el also turns out to be about one-half that for vulgare, which is expected from the lower net negative charge on E66 protein.