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.     

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.     

High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family

We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation. Received: 23 July 1997 / Accepted: 30 October 1997     

Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Distinction between light-mediated and light-independent variations in phytoplankton production rates

An array of factors simultaneously controls phytoplankton photosynthesis and hence the primary production process. Because their relative importance shifts both with depth and with season, the significance of individual factors cannot be resolved by in situ incubations, even if all relevant environmental and biotic variables are measured.Here a procedure is described by which in addition to in situ measurements, photosynthesis is simultaneously assessed in identical subsamples under constant temperature (10 °C) and light (0.66 mol m^–2 h^–1 PAR conditions, in vitro). By calculating photosynthesis per unit of chlorophyll, effects of shifting biomass on photosynthesis can be eliminated but seasonal variations of light-saturated photosynthesis generated by temperature, and vertical changes of light-requirements (e.g. by light-shade adaptation) remain obscure. Quotients of in situ photosynthetic rates divided by in vitro rates allow the quantification of light-mediated changes. Provided that photosynthesis measured in vitro is light-saturated, quotients in situ: in vitro rates should never exceed unity. They are a measure for the degree of light-limitation. In vitro rates normalized to chlorophyll give information on temporal changes caused by variations in photosynthetic capacity. In Lake Constance, mean cell size appears to control light-saturated assimilation numbers.