Febs Letters

doi: 10.1016/0014-5793(94)80104-5pmid: N/A

Coppola, Thierry; Waldmann, Rainer; Borsotto, Marc; Heurteaux, Catherine; Romey, Georges; Mattéi, Marie-Geneviève; Lazdunski, Michel

doi: 10.1016/0014-5793(94)80105-3pmid: 8307146

A cDNA encoding a N‐type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N‐type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N‐type Ca2+ channel gene has been localized on the chromosome 2, band A.

Ichiki, Yoshinari; Kitamura, Kazuo; Kangawa, Kenji; Kawamoto, Mari; Matsuo, Hisayuki; Eto, Tanenao

doi: 10.1016/0014-5793(94)80106-1pmid: 8307158

A specific and sensitive radioimmunoassay for human adrenomedullin has been developed and distribution and characterization of immunoreactive adrenomedullin in human tissue were investigated. The radioimmunoassay specifically recognizes its carboxyterminal region and half maximal inhibition of binding of radioiodinated adrenomedullin(40–52)NH2 was observed at 11 . Immunoreactive adrenomedullin was abundant in adrenal medulla (47.7 ± 26.1 , mean ± S.D.) and was ubiquitously found in all tissue examined. The mean plasma concentration of adrenomedullin in three normal individuals was 17.2 ± 6.4 (mean ± S.D.). By analysis with reverse‐phase high‐performance liquid chromatography coupled with the radioimmunoassay, most immunoreactive adrenomedullin in the adrenal medulla, atrium and lung was found to be adrenomedullin(1–52)NH2.

Sirangelo, Ivana; Bismuto, Ettore; Irace, Gaetano

doi: 10.1016/0014-5793(94)80107-Xpmid: 8307149

The stability of the acidic compact state of apomyoglobin toward the denaturant action of guanidinium hydrochloride and temperature was studied by examining the effects induced on the intrinsic tryptophanyl fluorescence and that of the adduct formed with 1,8‐anilinonaphthalenesulfonate (ANS). The results indicated that the disorganization of tryptophanyl environments is caused by a cooperative discrete molecular transition, thus contrasting the assumption that the acidic compact form of apomyoglobin might be a molten globule state. The unfolding of the ANS binding regions was found to involve, at least, two stages over a wide range of denaturant concentrations.

Howell, Mike; Shirvan, Anat; Stern-Bach, Yael; Steiner-Mordoch, Sonia; Strasser, Jane E.; Dean, Gary E.; Schuldiner, Shimon

doi: 10.1016/0014-5793(94)80108-8pmid: 8307150

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b‐VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N‐terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin>dopamine = norepinephrine>epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N‐terminus sequence of the purified vesicular amine transporter [Stern‐Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730‐9733].

Beltrán, C.; Darszon, A.; Labarca, P.; Liévano, A.

doi: 10.1016/0014-5793(94)80109-6pmid: 8307151

Ion fluxes through poorly understood channel‐mediated mechanisms participate in the interaction between spermatozoa and egg. Previously, we reported the characterization in planar bilayers of a high conductance Ca2+‐selective, voltage‐dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes [14]. Here we show that this ion channel can be directly transferred to planar lipid bilayers upon sperm addition, from sea urchin [S. purpuratus and L. pictus) and from mouse. We found that spermatozoa from these species posses a conspicuous Ca2+‐selective, high conductance, multi‐state, voltage‐dependent channel, which displays similar voltage dependence and equal PBa2+/PK + ~4 in the three species. The presence of this Ca2+ channel in such diverse species suggests it plays a relevant role in sperm physiology. The high sensitivity of planar bilayers to detect single ion channels can now be used to study ion channel regulation and gamete interaction.

Völkl, Harald; Busch, Gillian L.; Häussinger, Dieter; Lang, Florian

doi: 10.1016/0014-5793(94)80110-Xpmid: 7508402

Osmotic swelling of rat hepatocytes increases fluorescence of Acridine orange and of fluorescein isothiocyanate (FITC)‐dextran, both indicative of alkalinization of acidic intracellular vesicles. Similar to osmotic cell swelling, insulin and glutamine lead to an increase in Acridine orange fluorescence, an effect virtually abolished upon osmotic reversal of glutamine‐induced cell swelling. Barium, which blocks K+ channels in the plasma membrane, similarly leads to cell swelling and increase of Acridine orange fluorescence. Since proteolysis is governed by lysosomal pH, these observations indicate that the anti‐proteolytic action of osmotic cell swelling is mediated by lysosomal alkalinization. Thereby, insulin, glutamine and barium probably exert their anti‐proteolytic action by cell swelling and subsequent lysosomal alkalinization.

Bird, T.A.; Schule, H.D.; Delaney, P.; de Roos, P.; Sleath, P.; Dower, S.K.; Virca, G.D.

doi: 10.1016/0014-5793(94)80111-8pmid: 8307152

In KB cells, interleukin‐1 (IL‐1), epidermal growth factor and phorbol ester transiently activated both MAP kinase and a serine kinase which phosphorylated the heat shock protein hsp27. Extracts made from IL‐1‐stimulated KB cells phosphorylated recombinant hsp27, in vitro, on serine residues 78 and 82 which are contained within Arg‐X‐X‐Ser motifs similar to those phosphorylated by the ribosomal protein S6 kinases. Upon size exclusion chromatography, however, hsp27 kinase eluted as a single peak of activity at 50–60 kDa, clearly separated from ribosomal protein S6 kinases. Treatment of partially purified hsp27 kinase with protein phosphatase‐2a reduced its activity by 80%. De‐phosphorylated hsp27 kinase could be approximately 50% reactivated by a factor present in IL‐1‐treated cell extracts in the presence of ATP. This factor co‐eluted with MAP kinase after partial purification by DEAE‐cellulose, phenyl Sepharose, and size exclusion chromatography. Purified sea star p44mpk and recombinant ERK2 MAP kinases were also capable of re‐activating hsp27 kinase to a similar extent. These data suggest that hsp27 kinase is downstream from, and probably a direct target of MAP kinase.

Sutherland, Calum; Cohen, Philip

doi: 10.1016/0014-5793(94)80112-6pmid: 8307153

The α‐isoform of glycogen synthase kinase‐3 (GSK3α) was inactivated by 80% towards a synthetic peptide substrate upon incubation with Mg‐ATP and either MAP kinase‐activated protein (MAPKAP) kinase‐1 or p70 S6 kinase. Inactivation by either kinase resulted from the phosphorylation of Ser‐21 and was reversed by treatment with protein phosphatase 2A1. Phosphorylation also decreased GSK3α activity towards glycogen synthase, inhibitor‐2 and c‐jun. The specificity of GSK3a was similar to GSK3β, but with the synthetic peptide substrate heparin stimulated the dephosphorylated form of GSK3α (6‐fold) more than GSK3β(1.8‐fold). After phosphorylation, both isoforms were stimulated 15–20‐fold by heparin.

Maziere, Cécile; Auclair, Martine; Maziere, Jean-Claude

doi: 10.1016/0014-5793(94)80113-4pmid: 8307154

The effect of tumor necrosis factor on the oxidative modification of LDL by U937 human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation product content and the electrophoretic mobility of the particle. In the range of concentrations from 2.5 to 10 , the cytokine induced a dose‐dependent increase in cellular‐induced oxidation of LDL. This effect was accompanied by a stimulation of LDL degradation by J774 macrophage‐like cells. Concurrently, the TNF‐treated cells secreted superoxide anion with a higher rate. Since LDL oxidation is believed to be an inportant feature in the formation of the atherosclerotic plaque, the described effects of TNF might be of importance in long‐term exposure to this cytokine during inflammation.