Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.     

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.     

Early inductive interactions are involved in restricting cell fates of mesomeres in sea urchin embryos

Isolated intact caps of animal blastomeres, obtained from either 8- or 16-cell embryos, differentiate as swollen ectodermal vesicles. These findings agree with earlier studies demonstrating that mesomeres contribute only to larval ectoderm during normal development. In contrast, we find that pairs of mesomeres isolated from 16-cell embryos can differentiate endodermal and mesenchymal cells in a substantial number of cases (23%). Thus, mesomeres have a greater developmental potential than is realized during normal development. Further results support hypotheses that graded distributions of morphogenetic determinants exist within these embryos, since the extent of differentiation of isolated mesomeres is related to the relative position of the third cleavage plane along the animal-vegetal axis. When the third cleavage plane is subequatorial and the resulting animal blastomeres inherit a fraction of the vegetal hemisphere, more cases (39%) differentiate endodermal and mesenchymal cell types. A significant number of mesomere pairs (9-14%), however, can still differentiate endodermal and mesenchymal cells when the mesomeres are formed within the animal hemisphere. Thus, putative vegetal morphogenetic determinants may extend into the animal hemisphere in some cases. Further results indicate a temporal restriction in the developmental potential of mesomeres or mesomere progenitor cells since their differentiative capability is greater if they are isolated earlier during development. Aggregates of isolated mesomere pairs also display a decreased developmental potential when compared to isolated mesomere pairs. These results suggest that associations with adjacent cells (vegetal cells as well as adjacent mesomeres) restrict the development of mesomeres between third and sixth cleavages.     

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.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.