Protein kinase C activity in neonatal cultured rat cardiomyocytes supplemented with docosahexaenoic acid.

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after alpha 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the "in vivo" ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.     

P450-based porous silicon biosensor for arachidonic acid detection

A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 μM arachidonic acid in a dynamic range of 10-200 μM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 μM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 μM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.     

beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease.

The deposition of beta-amyloid in the brain is the key pathogenetic event in Alzheimer's disease. Among the various mechanisms proposed to explain the neurotoxicity of beta-amyloid deposits, a new one, recently identified in our and other laboratories, suggests that beta-amyloid is indirectly neurotoxic by activating microglia to produce toxic inflammatory mediators such as cytokines, nitric oxide, and oxygen free radicals. Three findings presented here support this mechanism, showing that beta-amyloid peptides (25-35), (1-39), and (1-42) activated the classical NADPH oxidase in rat primary culture of microglial cells and human phagocytes: 1) The exposure of the cells to beta-amyloid peptides stimulates the production of reactive oxygen intermediates; 2) the stimulation is associated with the assembly of the cytosolic components of NADPH oxidase on the plasma membrane, the process that corresponds to the activation of the enzyme; 3) neutrophils and monocytes of chronic granulomatous disease patients do not respond to beta-amyloid peptides with the stimulation of reactive oxygen intermediate production. Data are also presented that the activation of NADPH oxidase requires that beta-amyloid peptides be in fibrillary state, is inhibited by inhibitors of tyrosine kinases or phosphatidylinositol 3-kinase and by dibutyryl cyclic AMP, and is potentiated by interferon-gamma or tumor necrosis factor-alpha.     

Delayed larval development in <Emphasis Type="Italic">Anopheles</Emphasis> mosquitoes deprived of <Emphasis Type="Italic">Asaia</Emphasis>bacterial symbionts

BACKGROUND: In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi. RESULTS: Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic. CONCLUSIONS: The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.     

Alteration of macrophage surface in the course of immunological activation: decay of metabolic response to concanavalin A

“Activated” macrophages, isolated from lungs of BCG-infected rabbits, exhibited a greater perturbability of oxidative metabolism than their normal counterpart, when exposed in vitro to unrelated microorganisms or treated with phospholipase C. They were much less metabolically responsive when incubated with Concanavalin A. The metabolic effect of this lectin gradually decayed as time elapsed from the day of infection.These results are interpreted to show a modification of the surface properties of the macrophage in the course of the immunological activation. The possible mechanisms of generation of these surface alterations are discussed.     

Synthesis of adenine nucleotides from exogenous adenosine in the perfused rat heart under normoxic conditions and after ischemia

The rate of synthesis of myocardial adenine nucleotides from exogenous adenosine was studied in the isolated rat heart perfused under normoxic conditions and following ischaemia. The rate of incorporation of adenosine depended on the extracellular concentration of the precursor, following Michaelis-Menten kinetics with a apparent Km of 51.3 microM and a maximal rate of incorporation of about 1 100 nmol g-1 (wet wt.) 30 min-1. The adenosine uptake induced an increase in ATP concentration (+ 20%) when the exogenous concentration of precursor exceeded 10 microM. Following low-flow ischaemia (0.5 ml/min, 30 min), the rate of incorporation of 5 microM adenosine was diminished (-23%), but adenine nucleotide level restoration was favoured by the nucleoside administration. After total ischaemia (24 min), the extent of the decrease in adenosine incorporation was the same as in the case of moderate ischaemia but adenine nucleotide content was not restored.