Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.     

Micropatterned surfaces for the control of endothelial cell behaviour

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.     

Fibrinogen conformation and platelet reactivity in relation to material-blood interaction: effect of stress hormones

The performance of many biomaterials in hemocompatibility tests is altered when blood is drawn from stressed subjects. A salient physiological response during stress is one in which hormones are released into plasma by the hypothalamo-pituitary-adrenal axis. We investigated the influence of basal and stress levels of epinephrine and beta-endorphin on the conformation of fibrinogen (Fbg), both in saline solution (under physiological conditions) and after its adsorption to polyethylene (PE), by FT-IR spectroscopy. Moreover, as Fbg is one of the major mediators of platelet adhesion, the behavior of platelets in contact with PE was also evaluated as a function of the two different hormone concentrations. Epinephrine was found to affect Fbg conformation and to increase platelet adhesion to PE at stress level. Basal and stress levels of beta-endorphin did not significantly affect the Fbg conformation and only induced adhesion of isolated platelets to the PE surface. A direct relationship was therefore found between Fbg conformation and platelet behavior. The response of platelets was affected by the stress status of donors through the influence of epinephrine on Fbg conformation.     

Three highly oxygenated caryophyllene sesquiterpenes from Pestalotiopsis sp., a fungus isolated from bark of Pinus taeda

A Pestalotiopis sp. was isolated from the trunk bark of Pinus taeda. The fungus was cultivated in liquid medium and produced three highly oxygenated caryophyllene sequiterpene derivatives, named pestalotiopsolide A, taedolidol and 6-epitaedolidol, respectively. The sesquiterpenes were isolated by silica gel based chromatographic procedures and their structures were elucidated by NMR spectroscopic data.     

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells

2,3-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O₂ and nitric oxide.Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.     

A new mouse mutant for the LDL receptor identified using ENU mutagenesis

In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This loss-of-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality.     

Molecular cytogenetic study of three common Mediterranean limpets, Patella caerulea, P. rustica and P. ulyssiponensis (Archaeogastropoda, Mollusca)

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.