Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

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.     

Increased levels of c-fos and c-myc mRNA in ATP-stimulated endothelial cells

In bovine aortic endothelial cells, ATP induced a transient and sequential accumulation of c-fos and c-myc mRNA, which was detected after 1 hour and 3 hours, respectively. The effect of ATP on c-fos mRNA was stronger than that of TNF and bFGF. Both ATP and bFGF increased c-myc mRNA after a 3 hour treatment, whereas TNF did not. If none of the 3 agonists tested induced a selective expression of c-fos or c-myc, each of them was associated with a different quantitative combination of the 2 signals, which might be related to the distinct responses that they trigger in endothelial cells.     

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.     

In vitro effects of arachidonic acid and of inhibitors of its metabolism on the dog thyroid gland

Incubation of dog thyroid tissue with arachidonic acid (10 to 200 μM) led to the following events:

- low conversion to prostaglandins E₂ and F_2α: 0.07% and 0.02% per hour and 100 mg tissue, respectively

- inhibition of the stimulatory effect of low concentrations of TSH on thyroid secretion: the secretory effect of supra-maximal concentrations of TSH and of dB-cAMP was unaffected

- inhibition of the cyclic AMP accumulation induced by TSH: this effect was inhibited neither by indomethacin nor by ETYA; cyclic AMP accumulation in response to cholera toxin or PGE₁ was unaffected

- no effect on cyclic GMP level

- stimulation of thyroid proteins iodination.

ETYA, but not indomethacin, depressed the iodination of thyroid proteins in resting and stimulated tissue. These data show that arachidonic acid-or a metabolite-can modulate thyroid responsiveness to TSH and suggest that lipoxygenase-products of arachidonic acid metabolism could be involved in thyroid proteins iodination.     

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.