Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin

Protein kinase C (PKC) is a ubiquitous enzyme family implicated in the regulation of a large number of short- and long-term intracellular processes. It is hypothesized that modulation of PKC activity may represent, at least in part, a functional link between mutations (genotype) that lead to the pathological accumulation of naturally occurring compounds that affect PKC activity and perturbation of PKC-mediated substrate phosphorylation and cellular function in the corresponding diseases (phenotype). This model provides a unifying putative mechanism by which the phenotypic expression of some inborn errors of metabolism may be explained. Recent studies in a cell-free system of human skin fibroblasts support the hypothesis that alteration of PKC activity may represent the functional link between accumulation of sphingolipids and fatty acyl-CoA esters, and perturbation of cell function in sphingolipidoses and fatty acid oxidation defects, respectively. Further studies will elucidate the effects of these alterations on PKC-mediated short- and long-term cellular functions in these diseases, as well as the possible role of PKC in the pathogenesis of other diseases.     

Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages

Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.     

A sustained increase in the endogenous level of cAMP reduces the retinoid concentration required for APL cell maturation to near physiological levels

cAMP-dependent signal transduction co-operates with retinoids to induce acute promyelocytic leukaemia (APL) cell maturation. The rationale of this work was to determine whether signal cross-talk could be used to decrease the pharmacological doses of retinoids in the treatment of APL. When only the basal level of adenylate-cyclase (AC) activity is present in NB4 cells, up to 1 microM concentration of all-trans retinoic acid (RA) is required for full maturation (100%). In these conditions, with only 10 nM RA less than 20% of cells will differentiate. Although the use of membrane receptor agonists to activate AC has been proved to synergize with RA treatment, these agents were never as potent as cell permeant cAMP analogues. Analogues have disadvantages since cleavage by serum and cellular phosphodiesterases generates metabolites which interfere in cellular response. In the present study, we observed cell maturation by engrafting an autonomous Bordetella pertussis AC which steadily delivers natural cAMP into the cell. The enzyme alone had no effect on cell maturation. Importantly, cell maturation was increased in a dose-dependent manner when the bacterial AC (1 ng/ml to 1 microg/ml) was used to potentiate the effects of low doses RA (10 nM). More than 50% of cells matured with only 10 nM of RA and 200 ng/ml of B. pertussis AC. The maturation response was significantly increased when lower amounts of enzyme were repetitively added to the culture to compensate for enzymatic decay. These results indicate that a sustained AC activity enhanced cell maturation. We were able to reduce to 3 nM the RA requirement, provided that a minimal amount (20 ng/ml) of B. pertussis AC was added every 12 h in culture. Membrane signalling maintaining high the level of cAMP substantially improved the efficacy of APL cell maturation by retinoids. Therefore, therapeutic benefits are expected by lowering the concentration of RA towards physiological (nanomolar) levels, thus reducing the side-effects of the drug. cAMP-elevating drugs that act on a post-cyclase target (cyclic-nucleotide phosphodiesterases) or cell-targeted drug carriers (cAMP and RA loaded liposomes) should be evaluated as maturation therapies combining the activation of multiple signalling pathways.     

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A.1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.     

Effect of Nickel Nitrate Concentration on the Size of Nickel Oxide Nanoparticles Bio-synthesized by Artemisia herba-alba Aqueous Leaves Extract and Improving Their Antioxidant Activities

Journal of Inorganic and Organometallic Polymers and Materials - This work aims to investigate the effect of nickel nitrate concentration on the size and antioxidant activity of nickel oxide...     

Optimizing the Antibacterial Activity of Iron Oxide Nanoparticles Using Central Composite Design

This work aims to optimize the antibacterial activity of iron oxide nanoparticles (IONPs) against both Gram-positive and Gram-negative bacteria. IONPs were greenly biosynthesized using Moringa oleifera leaves extract, and surface methodology (RSM) based on central composite design (CCD) was employed to investigate the combined effect of various experimental factors on the antibacterial activity of IONPs. The reaction and annealing temperatures besides precursor concentration were set as independent variables, while the antibacterial activity was set as a response to obtain the optimal conditions that maximizes IONPs antibacterial activity. Different characterization techniques such as UV–Vis, FTIR, XRD, SEM, and EDX were employed to study the properties of the biosynthesized nanoparticles. Meanwhile, the antibacterial activity was tested using the disk diffusion method. The characterizations results have confirmed the biosynthesis of Hematite (α-Fe₂O₃) nanoparticles of rhombohedral structure. The generated model has exhibited predicted values very close to the actual proving its validity to analyze and optimize the studied process. The model indicated that all the investigated parameters and their interactions have significantly affected IONPs antibacterial activity. An optimal antibacterial activity was achieved when biosynthesis factors at their lower levels (? 1). Furthermore, the effect of IONPs size on the antibacterial activity was studied and the results shown that the latter is significantly related to the nanoparticles size.

Graphical Abstract