Induction of hormone receptor formation in the unicellular tetrahymena

Studies based on treatment with antibodies to thyrotropic hormone, luteotropic hormone, growth hormone or adrenocorticotropic hormone have shown that although the unicellular Tetrahymena does not possesssui generis receptors to all polypeptide hormones, such binding structures may arise, or become established in the membrane of the unicellular Tetrahymena in the presence of exogenous hormone. The Tetrahymena subjected to hormonal imprinting still contained an increased amount of hormone after six generation changes, which suggested that either hormone production had been induced by treatment, or the internalized hormone had not been degraded intracellularly. Thus the role of hormonal imprinting in receptor formation has also been substantiated by the immunocytochemical approach used in the present study.     

Effect of H-7 on the modulation of glucagon actions by activators of protein kinase-C

The stimulations of ureagenesis and cyclic AMP accumulation induced by glucagon were inhibited by 10 nM vasopressin or 100 nM phorbol 12-myristate 13-acetate (PMA). The maximal accumulation of cyclic AMP induced by glucagon was clearly diminished by these agents without change in the EC50 for the peptide hormone suggesting a non-competitive type of inhibition. H-7 blocked the inhibition of glucagon-stimulated ureagenesis induced by PMA and vasopressin and diminished their effect on the accumulation of cyclic AMP induced by glucagon. It is concluded that activation of protein kinase C inhibits the stimulation of ureagenesis and the accumulation of cyclic AMP induced by glucagon in liver cells from hypothyroid rats; H-7 inhibits the effects of protein kinase C activation.     

Functional alpha 2-adrenoceptors in rat adipocytes: inhibition of cyclic AMP accumulation

Alpha 2-adrenoceptor activation inhibits cyclic AMP accumulation in fat cells from many species. However, the presence of alpha 2-adrenoceptors in rat adipocytes has been difficult to demonstrate. We observed that alpha 2-adrenergic activation inhibits forskolin-stimulated cyclic AMP accumulation both in rat and hamster adipocytes; UK 14304, p-amino clonidine and clonidine were the agents with higher efficacy. The effect of UK 14304 was blocked by yohimbine but not by prazosin demonstrating the involvement of alpha 2-adrenoceptors. Pertussis toxin blocked the alpha 2-adrenergic effect. Our results demonstrate the presence in rat fat cells of alpha 2-adrenoceptors coupled to adenylate cyclase via "Gi".     

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.     

Spatial interactions in rapid pattern discrimination

We measured reaction times (RTs) for identification of a target among distracters under stabilized image conditions in which the positions of the target and the distracters were constant within a single experimental session. Under these conditions, the observer need not search for the target because its position is known. We nevertheless found that the presence of even a single distracter could elevate RTs. The magnitude of this effect depended on the distance of the distracter from the target and, for some observers, the distance of the distracter from the fovea. When we added not one but six background elements in a ring around the target, RT increased even more. If, apart from these neighboring distracters, the target was surrounded by more distracters located beyond the nearest neighbors, RT was, in general, not increased further. These findings suggest that adding background elements in a search task can elevate RTs in ways that are not dependent on the positional uncertainty of the target.     

Aliphatic hydrocarbons obtained from iron carbides: Possible stellar and meteoritic implications

Origins of Life and Evolution of Biospheres -     

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.     

Use of regularly structured bacterial cell envelope layers as matrix for the immobilization of macromolecules

Summary Carboxyl groups present on the outer face of the hexagonally ordered S-layer lattices from Bacillus stearothermophilus PV72 and Clostridium thermohydrosulfuricum L111-69 were activated with carbodiimide. The reaction of the activated carboxyl groups with free amino groups of low molecular weight nucleophiles was controlled by labelling with polycationized ferritin, a net positively charged topographical marker for electron microscopy, which densely binds to S-layers possessing free carboxyl groups. Carbodiimide-activated carboxyl groups were also allowed to react with amino groups of ferritin (MW 440 000) and invertase (MW 270 000). Covalent attachment of ferritin was examined by electron microscopy. Using invertase, approximately 1 mg enzyme was bound per mg S-layer protein indicating a high packing density of invertase molecules on the outer face of the S-layer lattice. The immobilized invertase retained 70% of its original activity.