Production of acetylcholine receptors from TE671 cells grown on microcarriers

Summary When grown in DMEM supplemented with 10 % fetal calf serum and using Cytodex 3® as microcarriers, TE671 cells entering the stationary phase optimally expressed acetylcholine receptors. These, receptors could be conveniently extracted from cell-saturated, microbeads or monodispersed cells obtained by trypsinization of microbeads. Typically, a 500 ml-batch gave 6–7 pmol of receptors which could be used as antigen to assay anti-acetylcholine receptor antibodies in the sera of myasthenic patients.     

Use of nitrilotriacetic acid (NTA) by Pseudomonas species through iron metabolism

Summary Nitrilotriacetic acid (NTA), when added to solid or liquid media, stimulated the growth of Pseudomonas strains, whereas other synthetic iron-chelators, such as ethylenediaminediacetic acid, ethylenediaminetetraacetic acid, ethylenediaminedihydroxyphenyl acetic acid or ethylene glycol-bis-(-aminoethyl ether)-tetraacetic acid, resulted in concentration-dependent growth inhibition. Experimental data such as stimulation of growth in iron-poor media, inhibitory effect on siderophore biosynthesis, promotion of iron-uptake by NTA, together with the inability of the Pseudomonas strains to use NTA as a carbon and/or a nitrogen source, demonstrated that NTA favours the bacterial growth of Pseudomonas through its scavenging properties for iron. Offprint requests to: J.-M. Meyer     

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

Raman spectroscopic study of the effects of Ca2+, Mg2+, Zn2+, and Cd2+ ions on calf thymus DNA: binding sites and conformational changes

The interaction of Mg2+, Ca2+, Zn2+, and Cd2+ with calf thymus DNA has been investigated by Raman spectroscopy. These spectra reveal that all of these ions, and particularly Zn2+, bind to phosphate groups of DNA, causing a slight structural change in the polynucleotide at very small metal: DNA (P) concentration ratio (ca. 1:30). This results in increased base-stacking interactions, with negligible change of the B conformation of DNA. Contrary to Zn2+ and Cd2+, which interact extensively with the nucleic bases (particularly at the N7 position of guanine), the alkaline-earth metal ions are bound almost exclusively to the phosphate groups. The affinity of both the Zn2+ and Cd2+ ions for G.C base pairs is comparable, but the Cd2+ ions interact more extensively with A.T pairs than Zn2+ ions. Interstrand cross-linking through the N3 atom of cytosine is suggested in the presence of Zn2+, but not Cd2+.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Uraniosomes produced in cultured rabbit kidney cells by uranyl acetate

Cultured rabbit kidney cells were exposed to uranyl acetate. This produced single-membrane-bound presumably lysosomal bodies (called 'uraniosomes') containing electron-dense crystals in the cultured cells. Similar crystalline deposits were seen in extracellular locations also. All uraniosomes and extracellular uranium deposits analyzed by electron-probe x-ray analysis were found to contain uranium, potassium, calcium and phosphorus. Traces of sulphur were detected in some but not all uraniosomes and extracellular uranium deposits.     

Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

Mating-Type Effect on CIS Mutations Leading to Constitutivity of Ornithine Transaminase in Diploid Cells of SACCHAROMYCES CEREVISIAE

Cis-acting regulatory mutations have been isolated that affect L-ornithine transaminase (OTAse), an enzyme catalyzing the second step of arginine breakdown in yeast. These mutations lead to constitutive synthesis of OTAse at various levels. Two different types of mutations have been recovered, both of which are tightly linked to the structural gene (cargB) for this enzyme. One type behaves as a classical operator-constitutive mutation similar to the cargB+O---1 mutation previously described (DUBOIS et al. 1978). The second type is peculiar in two respects: the higher level of constitutive OTAse synthesis and the expression of constitutivity in diploid cells. These mutations are designated cargB+Oh. They behave as usual operator-constitutive mutations in diploid strains homozygous for mating type (a/a or alpha/alpha), but the constitutivity is strongly reduced in a/alpha diploid cells.     

Presence de Chitinozoaires dans les calcaires siluro-devoniens de la Sierra Negra (Pyrenees centrales espagnoles)

A stratigraphical and paleontological study of the Silurian in the Benasque area of the high Pyrenees has made it possible to assign an Upper Pridolian age to certain limestones which occur near the top of a thick and monotonous serie of black shales. These limestones yeiled some remarkably well preserved chitinozoa together with some conodonts, orthocerases and pelecypods.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.