Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Effect of ethanol on the glucose-induced movements of protons across the plasma membrane of Saccharomyces cerevisiae NCYC 431

The study of glucose-induced proton fluxes in Saccharomyces cerevisiae NCYC 431 showed a decrease of proton net efflux by ethanol across the plasma membrane of energized cells. Furthermore a negative net proton efflux (an influx) occurred from a given ethanol concentration (between 1.3 and 1.5 M) whatever the experimental conditions used, thus allowing the definition of a nil-net exchange step where no net movement of protons across the plasma membrane could be observed. A new technique of ethanol tolerance determination in yeast based upon a correlation for the same ethanol concentration between both the collapse of the proton gradient and the growth cessation in cultures supplemented with ethanol after 8 h incubation was proposed. The defined method also showed a cumulated effect of temperature and ethanol on Saccharomyces cerevisiae NCYC 431.     

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.     

Effect of the fetal testis on the number of germ cells in the fetal ovary of rats in vitro

When ovaries from 13.5-day-old fetuses are explained and cultured in vitro for 4 days in a synthetic medium, the number of germ cells increases 6 fold, on average. This increase is only approximately 2 fold if a pair of 16.5-day fetal testes is cultured together with the ovaries or if the ovaries are cultured in a medium in which testes have previously been grown for 4 days. The effect of the latter medium persists if it is dialysed against fresh medium, which suggests that the conditioned medium contains one or several substance(s) of molecular weight superior to the cut-off of the membrane. The testicular effect seems to be effective mainly during the final phase of intense multiplication of the germ cells.     

Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor gamma.

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha, beta and gamma. Since RAR gamma is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RAR gamma selectivity.     

Effect of ethanol on membrane fluidity of protoplasts fromSaccharomyces cerevisiae andKloeckera apiculata grown with or without ethanol,measured by fluorescence anisotropy

Summary Direct measurements of membrane fluidity by fluorescence anisotropy of protoplasts fromKloeckera apiculata andSaccharomyces cerevisiae, a low and a high ethanol tolerant strain respectively, are presented. The comparison of the behaviour of the two strains grown with or without ethanol enabled us to demonstrate the existing relationship between ethanol tolerance and membrane fluidity.     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

Neonatal glutaric aciduria type II: An X-linked recessive inherited disorder

Summary A new case of neonatal glutaric aciduria type II is reported. Neonatal acidosis, hypoglycemia, and hyperammonemia were characteristic. The baby died at four days of age. Organic acid analysis revealed massive glutaric aciduria with elevated concentrations of butyric, isobutyric, n-butyric, and isovaleric acid in his urine. The baby's pedigree suggested strongly an X-linked recessive mode of inheritance. Clinically, biochemically, and genetically glutaric aciduria type II is an heterogeneous disorder. The neonatal form is an X-linked inherited disorder which presents early in life, and is associated with metabolic acidosis, hypoglycemia, and hyperammonemia, and leads to death in the neonatal period. The mild form is an autosomal recessive inherited disease which may present even in adults, and is associated with recurrent hypoglycemia without ketosis and usually improves. Nevertheless the same unusual organic acid pattern is observed in both forms. The basic biochemical defect must be distinct and has not been elucidated.     

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.