First Report of Arceuthobium vaginatum subsp. cryptopodum on Pinus mugo.

Southwestern dwarf mistletoe (Arceuthobium vaginatum (Willd.) Presl subsp. cryptopodum (Engelm.) Hawksw. & Wiens, family Viscaceae) is a serious and common pathogen of ponderosa pine (Pinus ponderosa Douglas ex Lawson & C. Lawson) in Colorado, Utah, Arizona, New Mexico, and northern Mexico (1). In July 2002, this dwarf mistletoe was observed parasitizing a 1.4-m tall mugo pine (P. mugo Turra) in the Black Forest north of Colorado Springs, CO (39°02.118'N, 104°36.028'W, elevation 2,250 m). The infected mugo pine was planted as an ornamental approximately 6 m from a ponderosa pine infected with A vaginatum subsp. cryptopodum. Dwarf mistletoe shoots were produced on the only infected branch observed but this was sufficient for a positive identification of the dwarf mistletoe. Although J. Weir successfully inoculated mugo pine with western dwarf mistletoe (A. campylopodum Engelm.) and lodgepole pine dwarf mistletoe (A. americanum Nutt. ex Engelm.) (2), to our knowledge, this is the first report of a dwarf mistletoe occurring naturally on P. mugo, as well as the first report of A vaginatum subsp. cryptopodum on P. mugo (1). Specimens of A vaginatum subsp. cryptopodum from P. mugo have been deposited in the Deaver Herbarium, Northern Arizona University, Flagstaff (Accession No. 73761). References: (1) F. Hawksworth and D. Wiens. Dwarf mistletoes: biology, pathology, and systematics. USDA Agric. Handb. 709, 1996. (2) J. Weir. Bot. Gaz. 56:1, 1918.

Effects of steroids on association of T-2 toxin with mammalian cells.

The effects of steroids on the association of T-2 toxin with cultured cells were evaluated. Preincubating cells with certain steroids led to a time- and concentration-related increase in total T-2-cell association. At maximally effective concentrations, the increase in association was 300-500%. This effect required a preincubation at 37 degrees C for a minimum of 10 min and was completely reversible after 20-30 min. Steroid treatment increased the rate of toxin-cell association and decreased the rate of dissociation. The effect was elicited by progesterone, estradiol, testosterone and diethylstilbestrol, but not by several other steroids tested. Binding of T-2 to isolated ribosomes was not altered by the steroids. We speculated that steroids somehow alter the state of ribosomal aggregation or assembly such that more toxin can bind after entering the cell.

Effects of emetine on the specific association of T-2 toxin with mammalian cells.

The effects of emetine on the association of T-2 toxin with Chinese hamster ovary cells were examined. T-2 toxin-cell association at both 4 degrees C and 37 degrees C was reduced by up to 90% after preincubation of cells with emetine. Emetine-induced reduction in T-2 toxin-cell association was time-, temperature-, and concentration-dependent. A 4-min preincubation with emetine at physiological temperature was required to develop the maximum inhibitory effect. After brief exposures (< or = 5 min), emetine's inhibitory effects on toxin-cell association were reversible. However, after longer exposure periods to emetine (60 min), toxin-cell association was irreversibly blocked. The addition of emetine to cells prebound with toxin resulted in dissociation at a rate 2 to 3 times slower than a competitive chase with nonlabeled toxin. Emetine did not compete directly for T-2 toxin binding to its receptor on isolated, purified, run-off ribosomes. However, the binding of toxin to purified ribosomes prepared from cells preincubated with emetine was markedly reduced. Scatchard analysis indicated that emetine's inhibitory effects on T-2 toxin-cell association were mediated through mixed allosteric and competitive types of inhibition at specific, intracellular, T-2 toxin ribosomal binding sites.

Effect of emetine on T-2 toxin-induced inhibition of protein synthesis in mammalian cells.

Chinese hamster ovary cells were used to examine the effect of emetine upon the toxicity of T-2 toxin and several related trichothecene inhibitors of polypeptide synthesis. Emetine inhibited protein synthesis and T-2 toxin-cell association in a concentration-dependent manner. The dose-response curves for these two effects were nearly identical. Over a narrow concentration range (0.3-3.0 micrograms/ml), emetine's inhibition of protein synthesis was partially reversible, whereas its inhibition of toxin-cell association was maintained for extended periods. This sustained inhibition of toxin-cell association, resulted in "desensitized" cells with reduced sensitivity to the inhibitory effects of T-2 toxin on protein synthesis. Similar results were obtained when emetine-preincubated cells were challenged with diacetoxyscirpenol, verrucarin A and roridin A. In contrast, there were no measurable effects of emetine upon the response of the cells to the less potent trichothecenes, deoxynivalenol, T-2 tetraol and verrucarol. In addition to emetine, several other inhibitors of polypeptide synthesis were examined for their effects on T-2 toxin-cell association and sensitivity to T-2 toxin. Of these, only cycloheximide inhibited toxin-cell association. Unlike emetine, sustained protection against the effects of T-2 toxin was not observed with cycloheximide.

Differential association of T-2 and T-2 tetraol with mammalian cells.

The interactions of T-2 and its metabolite T-2 tetraol (hereafter tetraol) with CHO (Chinese hamster ovary cells) and CHO ribosomes were studied. T-2 was about 300-fold more potent at inhibiting protein synthesis in CHO than was tetraol. Association of T-2 with CHO was highly specific and achieved a maximum at a concentration producing complete inhibition of protein synthesis. Association of tetraol with CHO was of low specificity, but the specific fraction did correlate with the dose-response curve for protein synthesis inhibition. Binding of both T-2 and tetraol to isolated CHO ribosomes was quantitatively similar and highly specific. With isolated ribosomes, each toxin competed effectively for the binding of the other. Using intact cells, tetraol competed for T-2 cell association, but not the converse. The kinetics at physiological temperature for total and specific T-2 cell association were much more rapid than those for tetraol. Furthermore, the rate of tetraol-cell association was indistinguishable from the rate for cellular uptake of tritiated water. At 0 degrees C, there was a substantial association of T-2 with cells, whereas none was observed with tetraol. The kinetics of dissociation of both toxins from CHO were similar. We conclude that T-2 rapidly crosses the cell membrane of cells and binds to the intracellular target, the ribosomes. In contrast, tetraol is taken up by the cell much more slowly, and many more toxin molecules are found in the cell than there are ribosomes. It would appear that the main physical property of the toxins that brings about these results is the relative hydrophobicities of the molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific association of T-2 toxin with mammalian cells.

The binding of radiolabeled T-2 toxin to a mammalian cell line derived from a Chinese hamster ovary (CHO) was studied. The toxin bound to, or was taken up by, cells in a time-, temperature- and concentration-dependent manner. The binding was saturable, of high affinity (Kd approximately 0.1 to 1 nM), reversible at 37 degrees (half-time approximately 2 hr), and specific. The kinetics of T-2-cell association and the rate of toxin-induced inhibition of protein synthesis closely paralleled one another. Likewise, the concentration-response for inhibition of protein synthesis and the toxin binding isotherm were similar. A synthetically derived epimer of T-2 bound less tightly to cells, but apparently to the same site as authentic T-2. The epimer was also less potent at inducing inhibition of protein synthesis. Two other trichothecene toxins, one more and one less toxic than T-2, blocked labeled T-2 binding to cells in a manner reflective of their protein synthesis inhibitory potencies. We conclude that the binding we defined is an accurate measure of the toxin responsible for inhibition of protein synthesis in CHO cells. The data also suggested that, at equilibrium, the interaction of T-2 with cells is not static, but is the sum of a continuous uptake and release process.

Binding of T-2 toxin to eukaryotic cell ribosomes.

The binding of radiolabeled T-2 to eukaryotic ribosomes was studied. The toxin bound to ribosomes in a time-, temperature- and concentration-dependent manner. The binding was saturable (0.3 nM), reversible at 37 degrees (half-time approximately 2.5 hr) and specific. The stoichiometry was one toxin molecule bound per ribosome. Binding of T-2 appeared to stablize the toxin recognition site to thermal degradation. A synthetically derived epimer of T-2 bound to the same ribosomal site as authentic T-2, but apparently with lower affinity. Two other trichothecene toxins tested blocked the binding of T-2 to ribosomes in a manner reflecting their protein synthesis inhibitory potencies. Anisomycin blocked the binding of T-2 to both isolated ribosomes and cells, whereas emetine blocked binding only to cells. Our data, together with that in the accompanying paper (Middlebrook JL and Leatherman DL, Biochem Pharmacol 38: 3093-3102, 1989), suggest that T-2 interaction with CHO cells is best viewed as a free, bidirectional movement of toxin across the plasma membrane and specific high-affinity binding to ribosomes.

Mitogenicity of formalinized toxoids of staphylococcal enterotoxin B.

Staphylococcal enterotoxin B is a potent mitogen for mouse and human lymphocytes. Mitogenic activity was retained after detoxification of the enterotoxin by formaldehyde at pH 5.0, 7.5, OR 9.5. The most active toxoid (pH 7.5) was separated into a monomeric, a dimeric, and a polymeric fraction (1 x 10(5) to 3 x 10(5) molecular weight) by gel filtration, and although each fraction demonstrated mitogenic activity, the polymeric fraction was clearly the most efficacious. These data show that mitogenicity of staphylococcal enterotoxin B does not depend on toxicity. This suggests that the mitogenic and toxic activities are effected by different sites on the molecule.

Evidence for cell-receptor activity in lymphocyte stimulation by staphylococcal enterotoxin.

A wide dose-response curve and the inhibitory effect on mitogenicity of specific antitoxin suggest that polyclonal lymphocyte activation by staphylococcal enterotoxin requires direct interaction of toxin with lymphocyte receptors of low avidity for the protein. Staphylococcal enterotoxins A, B, and C1 demonstrated equivalent mitogenic activity. Lymphocyte receptors involved in enterotoxin activation thus appear to be specific for nonantigenic regions of the toxin molecule. Monosaccharide (hapten) inhibiton data indicate that lymphocyte receptors for staphylococcal enterotoxin lack alpha-mannoside, galactose, acetylgalactosamine, acetylglucosamine, and fucose (or closely related saccharides) as determinant sugars and thus differ significantly in structure from lectin cell receptors.