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Two experiments examined the effect on the performance of Leghorn hens given diets containing flame-roasted grains from 140 to 490 d of age. In exp. 1, the inclusion of barley, corn and wheat roasted at 121, 119 and 149 °C, respectively, had no significant (P > 0.05) effect on productive performance. Experiment 2 contained a factorial arrangement of treatments that involved two sources of dietary barley (non-roasted or flame-roasted at 154 °C), two methods of milling (hammer or roller) and two dietary protein levels (15 or 18%). Hens given the diets that contained roasted barley had lower (P

Previous studies in human skin keratinocyte cultures have shown that sulfur mustard (HD) induces an immediate and irreversible increase in internal free calcium levels that was independent of external calcium concentrations. These findings suggested a role for calcium in the aetiology of HD-induced cell death and that modulation of intracellular calcium concentrations may assist in providing protection against this agent. In the current work, actively proliferating and confluent cultures of first passage neonatal human skin keratinocytes were used to assess the effect of altered intra- and extracellular calcium levels on HD toxicity. Treatment of cultures with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, or the calcium chelator BAPTA-AM, which reduce HD-induced elevation of intracellular free calcium, did not modulate the toxicity of HD. Furthermore, alteration of external calcium concentrations during these same experiments failed to elicit any change in the viability of HD-exposed cells. Treatment of confluent cultures with ionomycin at either low (100 μ m ) or high (1.2 m m ) external calcium concentrations also failed to modulate the toxicity of HD in any way. It appears that in neonatal human skin keratinocytes in culture, HD-induced intracellular calcium perturbation does not play a major role in HD-induced cytotoxicity.

Exposure of confluent cultures of human skin keratinocytes to sulfur mustard (SM) induced an immediate and irreversible rise in internal free Ca(2+) levels that was independent of external Ca(2+) concentrations. The response was rapid, beginning within 1min after addition of SM to the cells, and sensitive, with significant effects observed at 100 mum. The rise in [Ca(2+)](INT) was unaffected by zero external Ca(2+) but was blocked by prior incubation with thapsigargin. The sensitivity to and irreversibility of the effects of SM on Ca(2+) levels was paralleled by cellular toxicity as assessed using three different cell viability assays. In addition, the time course of the onset of irreversible toxicity in our cultures coincides with the time course of effects on [Ca(2+)](INT). SM was also found to displace specifically bound ATP from purinergic (P(2)) receptors. These results suggest that therapies aimed at protecting internal stores of Ca(2+) from disruption by SM, perhaps at P(2) receptors, may provide substantial benefit in protecting human skin cells from the toxic effects of this vesicant.

The hemocyanins of molluscs are aggregates of a cylindrical decameric subparticle that assembles into di-, tri-, tetra-, penta-, and larger multi-decameric particles with masses that are multiples of the 4.4 Md decamer. Electron micrographs of these hemocyanins typically show the particles with two profiles: circular representing the cylinder viewed from the end and rectangular representing the side-view of the hollow cylinder.The model proposed by Mellema and Klug from image analysis of a didecameric hemocyanin with the two decamers facing one another with collar (closed) ends outward fits the appearance of side-views of the negatively-stained cylinders. These authors also suggested that there might be caps at the ends. In one of a series of transmission electron microscopic studies of molluscan hemocyanins, Siezen and Van Bruggen supported the Mellema-Klug model, but stated that they had never observed a cap component. With STEM we have tested the end cap hypothesis by direct mass measurements across the end-views of unstained particles.

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.

Hemocyanins are copper containing, oxygen binding proteins found in many invertebrate species of the phyla Arthropoda and Mollusca. Molluscan hemocyanins are cylindrical macromolecular assemblies of a basic decameric unit. The hemocyanins of two chitons, Stenonlax conspicua and Mopalia muscosa. are decamers, while those of two gastropods, Fasciolaria tulipa and Pleuroplora gieantea. are di-decamers. The hemocyanins of a third gastropod species, Busvcon contrarium can exist in a spectrum of multi-decameric forms. Molecular weights of the various types of molluscan hemocyanins were measured by absolute light scattering and Scanning Transmission Electron Microscopy (STEM) as a first step in understanding multi-decamer formation in some types of hemocyanins.Hemocyanins were prepared as previously described, dialyzed into 1M HEPES buffer, pH 7.4, containing 0.05M MgCl and freeze-dried or negatively stained for STEM analysis. Specimens were examined at the Brookhaven STEM Biotechnology Resource, which was operated at 40 kV using a -140°C cold stage. The elastically scattered electron signal from the STEM large angle annular detector was used to form the images.

The hemocyanins of gastropods consist of aggregates of a cylindrical decameric subparticle that assembles into di-, tri-, tetra-, penta-, and larger multi-decameric particles with sedimentation coefficients of ca. 105 S, 130 S, 150 S, 170 S, and higher values. We are using STEM to measure the masses of individual particles and analytical ultracentrifugation to determine the distribution of sedimenting components.Hemocyanins were isolated from freshly collected hemolymph by gel filtration on BioGel A-5m columns. Samples were analyzed with schlieren optics in a Beckman Madel E ultracentrifuge. Specimens were diluted into 0.1 M HEPES, pH 8.0, 0.01 M MgC12 to a final concentration of 100 ug/mL and freeze-dried for STEM analysis. The STEM instrument was operated at 40 kV using a -140 °C cold stage. The elastically scattered electron signal from the STEM large angle annular detector was used to form the images. The specimens were imaged with 10 A pixels at a dose of 6-10 e/A2. Molecular weights of individual particles were measured as previously described.