Activation of human neutrophils by phorbol ester decreases the cytoplasm compactness and the lactoferrin content of the granulocytes.

Human neutrophils isolated from the blood were incubated in vitro for 30 min with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to study its effect on the fine structure of granulocytes and on the subcellular distribution of lactoferrin (Lf). Flow cytometry analysis of the human neutrophils showed that PMA induced a decrease in size and granularity of the cells. By electron microscopy, the PMA-treated cells showed numerous empty vesicles, smoothing of the cell surface, and a marked decrease in the compactness of the cytoplasmic ground substance.

Role of B and T lymphocytes in the specific immunosuppression induced by a protein released by porcine monocytes infected with African swine fever virus.

Some immunobiological aspects of host responses to an immunosuppressive protein (p36) released by porcine monocytes upon infection with African swine fever virus were analysed in a murine system. Treatment of normal, adult C57BL/6 mice with p36 (i) significantly delayed allogenic skin graft rejection; (ii) suppressed the specific plaque-forming cell response to immunization with heterologous erythrocytes; but (iii) induced marked increases in the numbers of 'background' splenic Ig-secreting plaque-forming cells. Cytofluorometric analysis of spleen cells revealed that a considerable fraction of all B cells, as well as CD4 and CD8 T lymphocytes, undergo blast transformation after p36 treatment.

1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A.

The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r.

Correlation between B-cell mitogenicity and immunosuppressor effects of a protein released by porcine monocytes infected with African swine fever virus.

Virus-free supernatants of cultured swine monocytes infected by African swine fever virus (ASFV) suppressed in vitro proliferation of porcine and human blood mononuclear cells in response to phytohemagglutinin and the in vivo primary immune response of C57BL/6 mice against sheep RBC. The supernatants were fractionated by discontinuous ion-exchange chromatography and subfractionated by double-step preparative isoelectric focusing. The pool of the most purified active subfractions (F5'EP-ASFV) is made up of heat-unstable material, can be stained by silver nitrate, and has an isoelectric point of 3.

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry.

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.

Analysis of the conformation and ligand-binding properties of the activation segment of pig procarboxypeptidase A.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All.

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases.

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.

Urea-gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products.

Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0-9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability.

Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A.

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods.

Kinetic differences of the calcium-binding protein in absorptive hypercalciuric renal stone formers.

Normal levels of 1.25-dihydroxycholecalciferol and different behaviour of the calcium binding protein kinetics, in a group of absorptive hypercalciuric stone formers indicate that intestinal calcium hyperabsorption in stone formers is due to an altered calcium transport at the intestinal level rather than a phosphate renal leak.

The activation segment of procarboxypeptidase A from porcine pancreas constitutes a folded structural domain.

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly alpha-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme.

The severed activation segment of porcine pancreatic procarboxypeptidase A is a powerful inhibitor of the active enzyme. Isolation and characterisation of the activation peptide.

The activation peptide of the monomeric procarboxypeptidase A from porcine pancreas was isolated by means of controlled trypsin digestion of the proenzyme followed by ion-exchange chromatography under dissociating conditions (7 M urea). The molecular weight of the isolated peptide was estimated to be around 11500-12000 (corresponding to approx. 100-103 residues) as judged by SDS electrophoresis and amino acid analysis, a figure that agrees with the differences between the corresponding values for procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino acid hydrolase, EC 3.