Structural organization of the 5' region of the thyroglobulin gene. Evidence for intron loss and "exonization" during evolution

More than one third of thyroglobulin (1190 residues out of 2750) is made of one peptide motif repeated ten times in tandem. Segments unrelated to the motif interrupt this structure at various places. The corresponding gene region, which extends over 40 x 10(3) bases, was studied in detail. All exon borders and exon/intron junctions were localized precisely and sequenced, and their positions were correlated with the repetitive organization of the protein. When intron positions were compiled on a consensus sequence of all repeats, three categories of introns were observed. Except between repeats numbers 5 and 6, an intron was invariably found within the Cys codon making the limit of each motif. This category of intron most probably reflects the serial duplication events responsible for the evolution of this region of the gene. All other introns, except no. 2, are found at positions were the repetitive structure is disrupted by "inserted" peptides. We present the hypothesis that this second category of introns was already present in the original unit before the first duplication. Thereafter, they would have experienced either complete loss (some units do not contain any intron) or partial or total exonization, resulting in the slipping of intronic material into coding sequence. Intron no. 2, finally, separates motif no. 1 at a position on the boundary between two segments presenting sequence homology. This last type of intron probably reflects an initial duplication event at the origin of a primordial thyroglobulin gene motif. With all these characteristics, the thyroglobulin gene is presented as a paradigm for the analysis of the fate of introns in gene evolution.     

VIP and related peptides induce rapid homologous desensitization in the human lymphoma SUP T1 cell line

Incubation of human SUP T1 lymphoblasts with VIP, helodermin and related peptides induced homologous desensitization within 5 min as indicated by: 1) a secondary decrease in cellular cyclic AMP levels, even in the presence of phosphodiesterase inhibitors, 2) a reduced capacity of cells to bind [125I]helodermin, 3) decreased helodermin stimulation of adenylate cyclase activity in membranes, and 4) unaffected NaF- and Gpp[NH]p-stimulated adenylate cyclase activities. The desensitizing ability of all peptides correlated with their efficacy to occupy cell receptors, except for [D-Phe2]VIP, a partial VIP agonist with low intrinsic activity, that did not desensitize.     

Isolation and primary structure of rat secretin

A major form of rat secretin was purified to homogeneity from small intestine, being detected with a porcine secretin radioimmunoassay throughout 7 chromatographic steps. The sequence of the heptacosapeptide amide H-S-D-G-T-F-T-S-E-L-S-R-L-Q-D-S-A-R-L-Q-R-L-L-Q-G-L-V-NH2 shows that rat secretin has a glutamine residue in position 14 instead of arginine as in pig secretin.     

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-triphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immmunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.     

Linear dichroism and orientational studies of carotenoid Langmuir-Blodgett films

The linear dichroism of single monolayers of lutein, zeaxanthin and a mixture of lutein and synthetic phosphatidylcholine has been measured. The angle of orientation of the carotenoid molecules was found to lie between 45° and 51° relative to the plane of the solid support. Although the adsorbed monolayers were mostly in a monomeric state, microscopic observations, as well as the II-A isotherms, indicated the existence of crystalline islets. The results have been interpreted in connection with Haidinger's polarization brushes.     

Methylation and expression of the human thyroglobulin gene

The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Effect of pH on binding of agonists and antagonists to rat heart muscarinic receptors.

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.     

Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.