L.Giorgio Roda

Positive and negative immunomodulation by opioid peptides

The data that follow review part of the existing evidence concerning the neuroimmune functions mediated by opioid peptides, with particular regard to dual immunomodulatory effects. Limited references to substances other than opioid peptides are included, mainly to emphasize the possible similarities in the mediation of neuroimmune interactions by different informational substances, while the interactions directed from the immune to the nervous system have deliberately been omitted.

A cell division-active protein from E. coli

A purification procedure for a protein obtained from an pathogenic strain of E. coli is described. The protein-called CNF-is active in inhibiting the duplication of cultured mammalian cells. Since nuclei division is apparently normal, treatment of cultured cells with CNF leads to the formation of gigantic, polynucleated cells. The purified protein is chromatographically and electrophoretically homogeneous. A partial characterization of CNF protein is also given.

Isolation and characterization of chromaffin granules from a pheochromocytoma (PC 12) cell line.

Abstract Chromaffin granules have been isolated from the pheochromocytoma clonal cell line, PC 12, The PC 12 cells, preloaded with radioactive catecholamines, were homogenized in isotonic sucrose and subjected to differential centrifugation and linear or stepwise sucrose gradient fractionation. Antigenic determinants characteristic of bovine chromaffin granules were detected both by immunofluorescence in intact cells and by complement fixation of isolated PC 12 granules. Electrophoretic analysis of the purified PC 12 granules revealed that the major proteins present have molecular weights similar to those of bovine chromaffin granule protein markers. The PC 12 catecholamine storage granules are thus suitable in vitro models for adrenal chromaffin granules.

Mechanisms of leu-enkephalin hydrolysis in human plasma.

The present work describes the kinetics of enkephalin hydrolysis by plasma enzymes and the fragmentation pattern of both the parent peptide and of the first hydrolysis by-products. The degradation kinetics were followed by positive identification of the hydrolysis fragments by chromatographic methods, by amino acid analysis and by scintillation counting of tritium-labeled enkephalin. In addition, the results presented confirm the role of the low molecular weight plasma components in the control of the hydrolysis of the peripherally-released enkephalins.

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.

Enkephalin activity on antigen-induced proliferation on human peripheral blood mononucleate cells

The effect of the two opioid pentapeptides met- and leu-enkephalin and of the tetrapeptide TYR-GLY-GLY-PHE on the stimulated proliferation of human peripheral blood mononucleate cells (PBMC) is described. Cell populations obtained from different donors were induced to proliferate with a Candida antigen. In the presence of the antigen, met-enkephalin and TYR-GLY-GLY-PHE had a concentration-dependent diphasic effect. The proliferation of PBMC was stimulated at high peptide concentration, while it was inhibited at the lower concentrations used. The effect of leu-enkephalin appears to be rather ambiguous, and directed towards the inhibition rather than the stimulation of PBMC proliferation.

Enkephalin-binding systems in human plasma.

Three amino acid-containing fractions present in human plasma are shown to bind both leu and met-enkephalin: serum albumin and two species of a much lower molecular weight, in all likelihood polypeptides. The amount of enkephalin associated with serum albumin seems comparatively smaller than that associated with the two low molecular weight systems. These systems jointly are apparently capable of binding a significant part of the circulating enkephalins. The possibility is suggested that the interactions described may play a role in maintaining the integrity of circulating enkephalins.

Control mechanisms of peripheral enkephalin hydrolysis in mammalian plasma.

The protection of the adrenal-released enkephalins from enzyme hydrolysis by endogenous plasma components was studied in laboratory animals and in man. The results indicate that mechanisms active in protecting leu-enkephalin from hydrolysis are present in the plasma of all species examined. The protection seems to be due to two groups of substances, possibly of peptidic nature. The amount of protection given by these substances seems to be sufficient to play a significant role in controlling the physiological levels of leu-enkephalin released into the bloodstream.

Hydrolysis and binding of leucine enkephalin to lymphomic and erythroleukaemic cell lines

Hydrolysis and binding of labelled leucine enkephalin have been measured in the presence of cell lines of lymphoid and erythroid origin. The radioactive label was found to be associated to all lines studied. In the presence of these cells, enkephalin is rapidly hydrolyzed, forming three tyrosine-containing peptides: Tyr, Tyr-Gly and Tyr-Gly-Gly. Conversely, the presence of selective protease inhibitors reduces both enkephalin degradation and binding. Data obtained suggest the involvement in enkephalin hydrolysis of aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In addition, they suggest that the radioactive label associated to cells can be related to the peptides formed by the enzyme degradation of enkephalin and not to the intact pentapeptide.

Interindividual variability of enkephalin-degrading enzymes in human plasma.

The interindividual variability of the hydrolysis of leucine enkephalin, and of the formation of its hydrolysis by-products has been studied in human plasma. In agreement with known data, the data obtained indicate that Leu-enkephalin is degraded by several enzymes, belonging to three classes: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The relative ratio of the substrate degraded by each enzyme class-as well as the expression of the single enzyme species within each class-appears to be individually determined. Interindividual variability observed seems controlled by two main factors: the pattern of enkephalin-degrading enzymes and, more notably, the low molecular weight plasma inhibitors. Both these factors appear to be partially specific of each donor. Possibly because of the composition of these factors, the hydrolysis pattern of the substrate is characteristic of each donor, and constant in blood obtained from successive drawings, at least within a relatively short period of time.