Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay

High-affinity binding sites for endothelin have been found in a human placenta membrane preparation. 125I-endothelin bound to placenta membranes at 20 degrees C with an association half-time of 30 min, whereas the binding was only slowly reversed with a dissociation half-time of 250 min. In saturation experiments, a single class of high-affinity binding sites was identified with an apparent dissociation constant (KD) of 24 pM and a maximal density of 240 fmol per mg of protein. The binding of 125I-endothelin was half-maximally inhibited by cold endothelin at a concentration (IC50) of 140 pM. In contrast, no inhibition was found at 10(-4) M for a variety of vasoactive peptides such as angiotensin II, vasopressin, neuropeptide Y, substance P, CGRP, bradykinin, leucine enkephalin or dynorphin A. Similarly, the binding was modulated neither by the calcium channel blockers nifedipine, verapamil or diltiazem, nor by the calcium channel agonist Bay k 8644. There was also no effect with the structurally-related bee venom apamin. Using this membrane preparation, endothelin-like activity could be measured in the medium of cultured human endothelial cells by competition binding technique.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids

Twomitochondrion-specific fluorochromes,10-N-nonyl acridineorange (NAO) and rhodamine 123 (Rh123), were used todetermine the mechanism responsible for alterations in energymetabolism of transformed rat embryo fibroblast cells isolated fromdifferent locations within multicellular spheroids. Accumulation ofRh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thusrepresents mitochondrial distribution only. A reproducible selectivedissociation procedure was used to isolate cells from differentlocations within the spheroids. After isolation, cells weresimultaneously stained with one mitochondrial stain and the DNA dyeHoechst 33342, and several parameters, including cell volume, weremonitored via multilaser-multiparameter flow cytometry. Our dataclearly show a decrease in the uptake of Rh123 in cells from theperiphery to the inner regions of the tumor spheroids, reflecting apersistent alteration in mitochondrial function. However, NAO stainingexperiments showed no reduction in the total mitochondrial mass perunit cell volume. Because cells were exposed to stain under uniformconditions after isolation from the spheroid, these data indicate thatdownregulation of mitochondrial function is associated with cellquiescence rather than a transient effect of reduced nutrientavailability. This result, which is in accordance with data from twoother cell lines (EMT6 and 9L), might reflect a general phenomenon inmulticellular spheroids, supporting the hypothesis that quiescent cellsin the innermost viable spheroid layer stably reduce theirmitochondrial function, presumably to compensate for lower nutrientsupply and/or decreased energy demand.

     

Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA

J.K. THOMPSON, K.J. MCCONVILLE, C. MCREYNOLDS AND M.A. COLLINS. 1997. Evidence is presented that linearized plasmid DNA is capable of electrotransforming Lactobacillus plantarum at a frequency 500-fold lower than with the covalently closed circular molecule. When the linearized plasmid was religated prior to transformation the transformation efficiency was < 10-fold higher, suggesting that open circular plasmid was only slightly more efficient in the transformation of Lact. plantarum than linear DNA. This observation has implications for direct cloning into this species since the high background transformation frequency produced by the linear DNA could potentially obscure the recovery of clones. Nevertheless, using positive selection for enhanced chloramphenicol resistance, cloned fragments of Lact. helveticus DNA were obtained using the shuttle vector pGKV110.     

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of membrane potential on divalent cation transport catalyzed by the "electroneutral" ionophores A23187 and ionomycin

Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.     

Metalloendoprotease inhibitors which block the differentiation of L6 myoblasts inhibit insulin degradation by the endogenous insulin-degrading enzyme

The cultured myoblasts of the rat skeletal muscle cell line L6 proliferate till confluency and then fuse to form myotubes and express a number of muscle-specific proteins. We had shown that this differentiation process is blocked by specific metalloendoprotease inhibitors. We now demonstrate that metabolizing L6 myoblasts and their cell extracts degrade insulin to acid-soluble fragments by a non-lysosomal pathway. About 90% of the insulin-degrading activity residues in the cytoplasm and is due to a 110-kDa enzyme known as the insulin-degrading enzyme. The same metalloendoprotease inhibitors that block the differentiation of L6 myoblasts also inhibit insulin degradation by the metabolizing L6 cells, their cell extracts, and the insulin-degrading enzyme immunoprecipitated from the cytosolic extracts by a monoclonal antibody. These results suggest that the insulin-degrading enzyme is the metalloendoprotease whose activity is required for the initiation of the morphological and biochemical differentiation of L6 myoblasts.     

Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.