Biological Modification of Trichothecene Mycotoxins: Acetylation and Deacetylation of Deoxynivalenols by Fusarium spp

Attempts were made to elucidate the acetyl transformation of novel trichothecene mycotoxins, 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one (deoxynivalenol) and its derivatives, by trichothecene-producing strains of Fusarium nivale, F. roseum, and F. solani. In the peptone-supplemented Czapek-Dox medium, F. roseum converted 3a-acetoxy-7a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (3-acetyldeoxynivalenol) to deoxynivalenol. 3-Acetyldeoxynivalenol was also deacetylated by intact mycelia of the three strains in sugar-free Czapek-Dox medium. The growing F. nivale acetylated deoxynivalenol to afford a small amount of 3-acetyldeoxynivalenol. 3a,7a,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (7,15-diacetyl-deoxynivalenol), which was then deacetylated to give 7a-acetoxy-3a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (7-acetyldeoxynivalenol). It was noted that the ester at C-7 was not hydrolyzed by the fungal mycelium.     

Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.     

Human operator dynamics in manual tracking systems with auditory input

Response characteristics of human operators in manual pursuit tracking with auditory input are investigated. The human operator hears in his left ear a sound whose frequency varies in proportion to an external random signal. At the same time, he hears in his right ear another sound whose frequency varies in proportion to the angle of a control lever of a potentiometer. The operator controls the angle of the lever so that the frequencies of the sounds in both ears remain as close as possible. The dynamics of the human operator is studied by assuming a manmachine system whose input is the external signal and whose output is the voltage of the potentiometer. A learning identification method proposed by one of the authors is used to calculate the weighting function of the man-machine system, which is displayed on a CRT screen in real time. During the tracking task, the skin potential activity (SPA) is measured as an index of arousal of the operator.     

A new pathway in the photoreaction cycle of trans-bacteriorhodopsin and the absorption spectra of its intermediates

The intermediates of trans-bacteriorhodopsin (trans-bR) in the photoreaction cycle were investigated under two different conditions. In a low salt and neutral pH medium (10 mM phosphate buffer, pH 6.6), trans-bR was irradiated with 500 nm light at –190 C, resulting in formation of batho-trans-bR (batho-bR^t). On warming in the dark, batho-bR^t converted to lumi-trans-bR (lumi-bR^t), meta-trans-bR (meta-bR^t) and finally to trans-bR. The intermediates N and O, which had been detected by others by flash photolysis, were not observed. The thermal decay of lumi-bR^t in a high salt and high pH medium (10 mM borate buffer with l M NaCl, pH 10.0) proceeded simultaneously through two pathways; one to meta-bR^t and another to trans-bR. About 72% of lumi-bR^t converted to trans-bR directly and the residue converted to meta-bR^t. By use of this value, the absorption spectra of batho-bR^t (_max: 626 nm), lumi-bR^t (_max: 543 nm) and meta-bR^t (_max: 418 nm) were calculated. A photoreaction cycle of bacteriorhodopsin was proposed on the basis of the above findings.     

Involvement of proteolytic acivity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.     

Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2.

Lymph nodes (LN) draining progressively growing tumors contain tumor-sensitized but not fully functional preeffector lymphocytes. These cells could acquire therapeutic efficacy and be expanded upon sequential culture with anti-CD3 mAb for 2 days followed by incubation in IL-2 for 3 days. Using the weakly immunogenic MCA 106 and MCA 205 murine sarcomas, we have further defined conditions of this anti-CD3/IL-2 activation with which preeffector cells differentiated into immune effector cells. In vitro activation and expansion of effector cells required sequential but independent stimulation with anti-CD3 and IL-2 because the simultaneous presence of both anti-CD3 and IL-2 at either stage did not enhance the efficacy of activation. Generation of effector cells by this two-stage activation was critically dependent on the optimal concentrations of anti-CD3 (1.0 microgram/ml) and IL-2 (2-10 U/ml). However, these conditions were not optimal for inducing the greatest cellular proliferation. In adoptive immunotherapy experiments, although the transfer of anti-CD3/IL-2-activated cells alone could mediate the regression of established metastases, the concomitant administration of IL-2 enhanced the in vivo activity of these cells. More importantly, tumor regression mediated by the anti-CD3/IL-2-activated cells was found to be immunologically specific. The specificity was determined by the tumor that stimulated the preeffector cell response. In spite of their in vivo antitumor effects, the anti-CD3/IL-2-activated tumor-draining LN cells did not exhibit detectable in vitro cytotoxicity against the tumor target in the 4-h 51Cr-release assay. In mice bearing progressive tumor, draining LN contained most preeffector cells. Some preeffector cells were also detected in the spleen whereas mesenteric LN did not demonstrate any reactivity. In kinetics studies, sensitization of preeffector cells in the draining LN occurred between 4 to 6 days after tumor inoculation. As the tumor progressed, the presence of preeffector cells declined gradually suggesting a tumor-induced suppression. These results define the conditions whereby tumor-draining LN cells could be stimulated, in the absence of tumor Ag, to develop into specific therapeutic effector cells. Our findings also raise the possibility of using similar approaches for isolating immune effector cells from cancer patients for adoptive immunotherapy.     

Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae.

By using the Cu2+ method (Y. Ohsumi, K. Kitamoto, and Y. Anraku, J. Bacteriol. 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined. Histidine and lysine in the medium expanded the vacuolar pool extremely. Glutamate also accumulated in the cells, but mainly in the cytosol. The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool. Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM. This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation. Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate. The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.