Domenico Romeo

Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain.

A novel protein family, showing a conserved proregion and a variable C‐terminal antimicrobial domain, and named0 cathelicidin, has been identified in mammalian myeloid cells. The conserved proregion shows sequence similarity to members of the cystatin superfamily of cysteine proteinase inhibitors. Cathelicidins are stored in the cytoplasmic granules of neutrophil leukocytes and release the antimicrobial peptides upon leukocyte activation. Some of these peptides can assume an α‐helical conformation, others contain one or two disulfide bonds, still others are Pro‐ and Arg‐rich, or Trp‐rich. In addition to bacterial killing, some of these peptides exert additional functions related to host defense such as LPS‐neutralization and promotion of wound healing.

Enzymatic basis of metabolic stimulation in leucocytes during phagocytosis: The role of activated NADPH oxidase☆

Abstract A comparison is made between the activities of KCN-insensitive NADPH- and NADH-oxidase associated with granules of guinea pig polymorphonuclear leucocytes. Both enzymes are activated when bacteria or polystyrene spherules are put in contact with the leucocytes. A rise in NADPH oxidase activity is already detectable after a few seconds from the addition of bacteria to leucocytes. The enhanced NADPH oxidase activity is due to a 10-fold decrease of Km, whose value approaches cellular NADPH concentration. Phagocytosis induces a 3-fold increase of the intracellular NADP + NADPH ratio, without affecting the NAD+ NADH ratio. In view of these results and other considerations, we conclude that the metabolic stimulation of PMN leucocytes by phagocytosis is supported by changes in kinetic properties of NADPH oxidase.

Surface‐reactive stimuli selectively increase protein phosphorylation in human neutrophils

~ospho~iation of proteins is an important regulatory mechanism in metabolic pathways [I]. A sudden change in the turnover of some membrane phosphoproteins can be the critical event in the coupling of stimulation by extracellular ligands with appropriate cell response [2]. A coordinated and transient rise in the activity of protein kinase(s) and phosphatase(s) may thus represent one of the key mechanisms of regulation of the functions of cells responsive to external stimuli. One such cell is the blood neutropll~. In fact, exposure of neutrophits to a variety of particulate as well as surface-reactive soluble stimuli (formyl-peptides, phorbol esters)elicits a complex set of metabolic and functional changes. These include activation of O2 reduction to 0; [3-S], a transient rise in cyclic AMP concentration [6-81 and in protein methylation [9], translocation of calcium pools ]lO], Mottola and D. R., in preparation), enhanced phospholipid turnover [ 11 ,I 23, and activation of random and oriented locomotion [7-10,13-161 andofsecretion [17-191, Thus, neutrophils present an interesting system with which to investigate protein phosphorylation as an intracellular regulatory mechanism. We present here evidence that interaction of 32Plabeled human neutrophils with known activators of their functions, such as N-formyl-methionyl-leucylphenylalanine (FMLP) and phorbol 12-myristate-13acetate (PMA) [4,5,8 JO,1 3 ,15 ,I 6 ,191, results in an immediately increased incorporation of 32P into

Successful Therapy Of Niemann-pick Disease By Implantation Of Human Amniotic Membrane

In a patient with a lysosomal storage disorder, not involving the CNS, repeated implantations of human amniotic sheets have proved to provide a successful approach to enzyme replacement therapy. Implantation of pure epithelial cells, separated from the other cell types of the amnion, might markedly improve the procedure, avoiding some risks of host-versus-graft rejection.

Chemotactic and protease‐inhibiting activities of antibiotic peptide precursors

We have recently shown that two antimicrobial peptides (Bac5 and Bac7) and/or their immature forms (proBac5 and proBac7) can be released extracellularly from activated neutrophils. In the present study we have investigated the biological activities of the immature forms, which do not exhibit antimicrobial effects. We show that proBac7 is a monocyte‐selective chemoattractant, potentially contributing to the recruitment of these cells to infection sites, whereas proBac5 efficiently inhibits the in vitro activity of cathepsin L, a cysteine proteinase thought to contribute to tissue injury in inflammation.

The Cathelicidin Family of Antimicrobial Peptide Precursors: A Component of the Oxygen‐Independent Defense Mechanisms of Neutrophilsa

Host protection from invading pathogens involves cellular and humoral effectors and results from the concerted action of both non-adaptive (innate) and adaptive (acquired) immunity. The latter is based on specific immunological recognition mediated by clonally distributed receptors, is a recent acquisition of the immune system, and is present only in vertebrates. The former evolved well before the development of adaptive immunity and consists of a variety of cells and molecules distributed

Biochemical Properties of Bovine Granulocytes

Summary Granulocyte fractions, containing an average of about 92% neutrophils, were isolated from bovine blood. The electron microscope observation of these fractions showed that neutrophil granules have different shapes, but are all highly and homogeneously electron-dense. With respect to the granulocytes of human blood, bovine cells appear to have a lower content of azurophil enzymes and virtually lack lysozyme. Lysates of bovine granulocytes efficiently kill both E. coli (at pH 6.0 and 7.4) and S. aureus (mainly at pH 7.4). When exposed to opsonized B. mycoides, intact bovine granulocytes exhibit a marked enhancement in oxygen consumption, generation of O2 - and H2O2, and glucose oxidation through the hexose monophosphate pathway. About 15% of the total oxygen reduced is recovered extracellularly as O2 -. Hydrogen peroxide generated by phagocytizing cells is only partially utilized in reactions catalyzed by catalase and myeloperoxidase, and appears to mainly enter the glutathione cycle.

Activation of protein kinase C in neutrophil cytoplasts: Localization of protein substrates and possible relationship with stimulus-response coupling

Treatment of enucleated, granule‐free neutrophil cytoplasts with the protein kinase C activator phorbol 12O‐myristate‐13‐acetate (PMA) causes an increased ‐32P‐incorporation into a variety of polypeptides. Permeabilization of PMA‐stimulated, 32P‐labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA‐induced activation of generation of Superoxide anion (O− 2) and the phosphorylation of a cytosolic polypeptide with an apparent M r, of 46000, whose ‐32P‐labeling is also enhanced by the treatment of cytoplasts with 1‐oleyl‐2‐acetylglycerol, the Ca2+ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the 32P‐labeling of proteins in the 40000 M r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O− 2 production by PMA.

Lysosomes in heart tissue

Abstract 1. 1. The acid hydrolase (β-glucuronidase, EC 3.2.1.31; cathepsin; β-galactosidase, EC 3.2.1.23; acid ribonuclease, EC 2.7.7.17 and acid deoxyribonuclease, EC 3.1.4.6) of beef-heart muscle are associated with cytoplasmic particles and are poorly reactive towards external substrates; however, their activity is enhanced or even fully displayed by injuring the particles with a variety of treatments (osmotic shock, prolonged homogenization, alternate freezing and thawing, addition of Triton X-100). 2. 2. By gradually increasing the concentration of Triton X-100 in the enzyme assays, the acid hydrolases are gradually liberated from the particles, although not in equal proportions. 3. 3. When particles in suspension are submitted to the action of various “protein” or “lipid” reagents, it appears that both types of reagent are able to increase the availability of the hydrolases for their substrates, but that a high solubilization of the enzymes is achieved only by attacking the membrane phospholipids. 4. 4. Preincubation either in hypo- or hypertonic sucrose solutions produces an increase in the availability of the hydrolases; under the same conditions, a partial solubilization, albeit to a different extent for each enzyme, is also observed. 5. 5. Each of the enzymes considered displays a peculiar intracellular distribution pattern, but none of them is strictly the same as that of succinoxidase. By isopicnic centrifugation of a mitochondrial fraction it is possible to isolate a particulate fraction ( d = 1.174) showing a high concentration of latent hydrolytic enzymes and a comparatively low concentration of cytochrome c oxidase. 6. 6. The acid hydrolases of beef heart do not seem, therefore, to belong to mitochondria but to exhibit the main features of lysosomal enzymes. A comparison between the properties of beef-heart and rat-liver lysosomes is also made and the heterogeneity of the former is discussed.

Molecular cloning of Bac7, a proline- and arginine-rich antimicrobial peptide from bovine neutrophils

Bac7 is a 7 kDa proline‐ and arginine‐rich antimicrobial peptide which was purified from bovine neutrophils. We have used PCR to clone the cDNA of Bac7 precursor, a polypeptide of 21,569 Da. This cDNA is highly conserved in the 5′ region, with respect to the corresponding region in the precursors of several other structurally unrelated myeloid antimicrobial peptides. Furthermore, a 148 nt non‐coding region at the 3′ end is 75% homologous to a corresponding region of the cDNA of the precursor of PR‐39, a porcine antibacterial peptide which is also proline‐ and arginine‐rich.