Purinergic modulation of cardiac activity in the flounder during hypoxic stress

Summary The interaction of adrenaline and adenosine was examined in cardiac tissue of the flounderPlatichthys flesus.When applied alone both agents increased contractility in both auricular and ventricular myocardial strips. This positive inotropic effect was associated with a small depolarization in the tissues examined by the sucrose gap technique. Simultaneous application of adrenaline and adenosine gave an inhibition of the control responses seen with either agent alone in both auricle and ventricle.Radiocalcium flux studies on ventricular tissue showed that influx was increased by adrenaline or adenosine alone above control values, but when applied together radiocalcium influx was reduced. Radiocalcium efflux from cardiac microsomes was stimulated by challenge with adrenaline or adenosine alone. This stimulation was not seen following simultaneous challenge by both agents.The effect of adrenaline on responses of hypoxic flounder hearts was less than that seen in normoxic hearts. This situation was reversed by pretreatment with the purine receptor blocker caffeine. Caffeine pretreatment also reduced the positive inotropic effect seen in normoxic hearts challenged with adenosine.TLC studies gave strong evidence that hearts perfused with hypoxic salines produced both adenosine and adrenaline.The results are discussed as evidence for a mechanism of heart regulation which the flounder may use as a defence against severe acute hypoxic stress.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: I. Process concept and enzyme binding studies for pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase.     

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.     

Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin

An iodinated azido derivative of ketanserin, 7-azido-8-[125I]iodoketanserin ( [125I]AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) [125I]AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of [3H]dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of [125I]AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of [125I]AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that [125I]AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.     

Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter

The binding of [3H]reserpine ([3H]RES) to purified bovine chromaffin granule membranes has been studied at low membrane concentration. Saturation isotherms indicated a dissociation equilibrium constant KD of 30 pM and a density of binding sites of 8 pmol/mg of protein at 30 degrees C. The association rate constant was 4.0 X 10(5) M-1 s-1, and the calculated dissociation rate constant was 1.2 X 10(-5) s-1, corresponding to a half-lifetime of about 16 h. Although this dissociation was too low to be measured directly, [3H]RES binding was indeed reversible since it was lost after addition of the detergent Triton X-100. Dihydrotetrabenazine (TBZOH) inhibited [3H]RES binding in a time-dependent manner, EC50 varying from 37 nM after a 1-h incubation to 600 nM after 16 h. On the contrary, [3H]RES binding inhibition by the substrate noradrenaline was time independent. It is proposed that the transporter exists in two different conformations which bind exclusively either tetrabenazine (TBZ) or RES and which are in equilibrium. The effects of detergents were consistent with this two-conformation model. The transporter solubilized by cholate bound [3H]TBZOH, but not [3H]RES. On the other hand, addition of cholate to membrane-bound [3H]RES solubilized the membrane without releasing the ligand from its binding site. It is proposed that the TBZ-binding conformation is obtained by solubilization with cholate and that RES stabilizes the RES-binding conformation, allowing its solubilization by this detergent.     

Absorption, translocation, and metabolism of14C-clomazone in soybean (Glycine max) and threeAmaranthus weed species

The patterns of clomazone (2-[(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxazolidinone) absorption, translocation, and metabolism and their contribution to the plant selectivity of this herbicide were studied in tolerant soybean [Glycine max (L.) Merr.] andAmaranthus hybridus and in susceptibleA. retroflexus andA. lividus. Differential root absorption appeared to play a significant role in the differential response of these four plant species to clomazone. Absorption of root-applied¹⁴C-clomazone was greater by the two sensitiveAmaranthus weeds than by the tolerant soybean andA. hybri-dus. Following application of¹⁴C-clomazone to roots, most of the absorbed radioactivity was translocated to the leaves of all four species. Approximately 50% of the absorbed¹⁴C-clomazone was metabolized by all four plant species as early as 12 h after treatment. Thin layer Chromatographic (TLC) analysis of plant tissue extracts from all four species revealed the formation of two major metabolites of clomazone. These unidentified metabolites had Rf values of 0.4 and 0.8, respectively, in a butanolacetic acidwater (1235, vol/vol/vol) developing system. The Rf value of unaltered clomazone in this system was 0.95. Differential metabolism or differential rate of metabolism of clomazone was not observed in this study and did not seem to account for the tolerance of soybean andA. hybridus or the suceptibility ofA. retroflexus andA. lividus to this herbicide.Plant Pathology, Physiology, and Weed Science Department, Contribution No. 600.     

Primary Infection of Barley by Erysiphe graminis f. sp. hordei in Relation to Leaf-Age Dependent Resistance and the Roles of the Epidermis and Mesophyll in this Resistance

Abstract The infection frequency of both compatible and incompatible races of Erysiphe graminis f. sp. hordei decreased gradually with increasing leaf age on undetached primary barley leaves. The length of secondary hyphae of the compatible race was approximately the same regardless of age, but secondary hyphae were slightly longer on younger seedlings than on older seedlings in the case of the incompatible race. Both the infection frequency and length of secondary hyphae of the two races weredistinctly different. On composite sections produced by exchanging the epidermal layers of young and relatively mature primary leaves, the infection frequency of the compatible race was higher on the epidermis of young leaves than on the epidermis of older, leaves, regardless of which mesophyll was under the epidermis. The epidermis appears to play a major role in age-dependent resistance, while the mesophyll may act disparately by providing a factor promotive to mildew infection in addition to supporting the resistance function of the epidermis.