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.     

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.     

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.     

Retinoic acid application to chick wing buds leads to a dose-dependent reorganization of the apical ectodermal ridge that is mediated by the mesenchyme

Local application of retinoic acid to wing buds of chick embryos leads to dose- and position-dependent changes in the pattern of cellular differentiation. Early effects of retinoid treatment on the apical ectodermal ridge coordinate pattern changes and morphogenesis. The length of the apical ridge increases when additional digits will form but decreases when digits are lost. These changes in length can be understood in terms of a threshold response to the local retinoid concentration that results in either disappearance or maintenance of the ridge (Lee & Tickle, J. Embryol. exp. Morph. 90, 139-169 (1985)). Here, we have analysed the mechanisms involved in ridge disappearance by locally applying retinoic acid to the apex of stage 20 chick wing buds. With this treatment regime, low doses give duplicated digit patterns and higher doses truncations. The height of the apical ridge is progressively reduced with increasing doses of retinoid and the time course of ridge flattening indicates that the height of the ridge is correlated with bud outgrowth. With high doses of retinoic acid, the typical ridge, a pseudostratified epithelium in which the columnar cells are tightly packed, disappears and the epithelium at the tip of the bud consists of loosely packed cuboidal cells. Shortly after treatment, there is a decrease in the number of gap junctions between ridge cells. This early change in cell contacts suggests that gap junctions may be involved in maintaining epithelial morphology. When treated epithelium is recombined with untreated mesenchyme, an apical ridge is reestablished and distal structures can be generated. In contrast, when treated mesenchyme is recombined with the epithelium from normal buds, only proximal structures are formed. Therefore, retinoids can lead to a reorganization of the apical ectodermal ridge which is mediated and maintained by the mesenchyme.     

Field cage performance of two tachinid parasitoids of the tomato fruitworm on insect resistant and susceptible tomato lines

Rates of parasitism ofHelicoverpa (=Heliothis) zea (Boddie) (Lepidoptera: Noctuidae) by two tachinid parasitoid species differing in larviposition habits were measured in field cages on tomato plant lines with or without methyl-ketone (2-tridecanone and 2-undecanone)-mediated insect resistance. Hosts were placed on resistant or susceptible plants, exposed to parasitoids for 24–48 h, then held on artificial diet for parasitoid emergence. Rates of parasitism byArchytas marmoratus (Townsend) (Diptera: Tachinidae), which larviposits on its host's food plant, were significantly reduced on the resistant plants, relative to those on the susceptible plants. Parastism by another Tachinid,Eucelatoria bryani Sabrosky, which larviposits directly into its host and does not directly contact the foliage of its hosts' food plant as a small larva, was not affected by the methyl-ketone mediated resistance.     

Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.