Interaction between cystatin-derived peptides and papain

The interaction between papain and synthetic peptides which tentatively mimic cystatin surfaces was investigated both enzymatically and structurally. Measurements of dissociation equilibrium constants for the interaction of papain with these peptides modified by successive deletions or substitutions demonstrated that the QVVAG segment, which is highly conserved throughout members of the cystatin superfamily, is essential for the interaction. The glycylcontaining (N-terminal) fragments and PW-containing (C-terminal) fragments were found to be of lesser importance, since each could be deleted without significantly modifying the interaction. These fragments improved the stability of the interacting QVVAG region, which appeared to be substrate-like in all peptides tested, as it was cleaved at the A-G bond upon peptide-papain interaction. Replacement of the A residue at the scissile bond of the QVVAG by a blocked cysteinyl residue reduced the rate of cleavage of the susceptible bond and therefore shifted the resulting peptide from a substrate to an inhibitor. Derivatization of this substituted peptide at its N- and C-terminal ends by fluoresceinyl groups resulted in a dramatic decrease in theK _i to 0.5 µM. This improvement in the inhibitory properties of the substituted and derivatized peptides was correlated with structural changes as analyzed by molecular dynamic calculations. The results were compared to those proposed for the mechanism of inhibition by natural inhibitors of the cystatin superfamily.     

The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid

Abstract: Using a new high performance liquid chromatographic method we have measured tryptophan, 5-hydroxyindoleacetic acid (5HIAA), indoleacetic acid (IAA), and indolepropionic acid (IPA) in rat and human CSF. Experiments on rats indicate that IPA in CSF is not derived from the CNS but from bacterial metabolism in the intestine. However, IAA in CSF is derived from CNS tryptamine metabolism. Some tryptamine that is formed peripherally diffuses across the blood-brain barrier and augments the tryptamine formed within the CNS. We have concluded from our data that (i) measurements on CSF are a useful way of studying trace amine metabolism in human CNS, but it is essential to establish the anatomical and metabolic origin of any metabolite found in the CSF; and (ii) tryptamine metabolism is more important in man than in the rat.     

Beta-adrenergic influences on the firing of raphe dorsalis neurons after neonatal noradrenergic deafferentation in the rat

In Rat, neonatal lesion of the locus coeruleus induces modifications of beta-adrenergic regulations at the level of raphe dorsalis neurons. In normal conditions, the firing of serotoninergic raphe cells is not beta-adrenergic dependent. On the contrary in the lesioned animal, iontophoresis of a beta-blocking agent (DL-propranolol) produces a marked inhibition of the serotoninergic unit firing. Various mechanisms may account for this pharmacological response in the lesioned group: a classical hypersensitivity phenomenon, or the persistence of an immature regulation.     

Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment

The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [³H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measued. The specific radioactivity of the secertory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins is proposed.     

Rapid binding of beta 2-microglobulin to renal brush-border membranes

125I-labelled human beta 2-microglobulin binding to rat renal brush-border membranes was assessed by an in vitro assay under near physiological incubation conditions (i.e. low content of albumin). Binding rate was 55 pmol/min per mg protein in the presence of 200 nM of beta 2-microglobulin and degradation rate was negligible versus binding rate. The binding rate was in reasonable agreement with the in vivo reabsorption rate, supporting the hypothesis of proteins binding to the luminal membrane during the process of reabsorption. Mild solubilizing treatment (Triton 0.1%) of brush border after beta 2-microglobulin binding yielded the labelled molecule associated with a high-molecular-weight component. Aminopeptidase activity and binding ability were to a certain extent co-purified during the course of the brush-border preparation, suggesting that most of the beta 2-microglobulin binding sites were localized in the brush-border membranes.     

QTL Dissection of Lag Phase in Wine Fermentation Reveals a New Translocation Responsible for Saccharomyces cerevisiae Adaptation to Sulfite

Quantitative genetics and QTL mapping are efficient strategies for deciphering the genetic polymorphisms that explain the phenotypic differences of individuals within the same species. Since a decade, this approach has been applied to eukaryotic microbes such as Saccharomyces cerevisiae in order to find natural genetic variations conferring adaptation of individuals to their environment. In this work, a QTL responsible for lag phase duration in the alcoholic fermentation of grape juice was dissected by reciprocal hemizygosity analysis. After invalidating the effect of some candidate genes, a chromosomal translocation affecting the lag phase was brought to light using de novo assembly of parental genomes. This newly described translocation (XV-t-XVI) involves the promoter region of ADH1 and the gene SSU1 and confers an increased expression of the sulfite pump during the first hours of alcoholic fermentation. This translocation constitutes another adaptation route of wine yeast to sulfites in addition to the translocation VIII-t-XVI previously described. A population survey of both translocation forms in a panel of domesticated yeast strains suggests that the translocation XV-t-XVI has been empirically selected by human activity.