Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: critical roles for P-selectin glycoprotein ligand-1 and heterotrimeric G(i)-linked receptors

Lymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. We developed a novel intravital microscopy model to directly analyze through the skull the interactions between lymphocytes and the endothelium in cerebral venules of mice. No adhesive interactions were observed between lymphocytes and the nonactivated endothelium in the cerebral microcirculation. When brain venules were activated by pretreating mice with TNF-alpha or LPS, proteolipid protein 139-151 autoreactive T lymphocytes rolled and arrested; notably, only a few peripheral lymph node cells rolled and firmly adhered. Abs anti-P-selectin glycoprotein ligand-1 and anti-E- and P-selectin blocked tethering and rolling of autoreactive lymphocytes, suggesting that P-selectin glycoprotein ligand-1/endothelial selectins are critical in the recruitment of lymphocytes in inflamed brain venules. E- and P-selectin were expressed on cerebral vessels upon in vivo activation and had a patchy distribution during the preclinical phase of active and passive experimental autoimmune encephalomyelitis. LFA-1/ICAM-1 and alpha(4) integrins/VCAM-1 supported rolling, but were not relevant to rolling velocity. Firm arrest was mainly mediated by LFA-1 and ICAM-1. Pretreatment of autoreactive lymphocytes with pertussis toxin blocked integrin-dependent arrest, implicating a requirement for G(i) protein-dependent signaling in vessels from nonlymphoid districts. In conclusion, our data unveils the molecular mechanisms controlling the recruitment of autoreactive lymphocytes in inflamed cerebral vessels and suggest new insights into the pathogenesis of autoimmune inflammatory diseases of the CNS.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores

Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.     

Pancreatic polypeptide-related tumors

PP-producing tumors are mostly located in the pancreas and may present as three pathologic lesions: pure PP-omas, mixed tumors with minor PP cell population, and PP-cell hyperplasia. These tumors are among the most common multiple adenomas frequently found in patients with multiple endocrine neoplasia type 1. Hypersecretion and high circulating levels of PP are frequently found. They are symptomless but may be useful for the identification of the pancreatic tumors. Numerous types of extrapancreatic endocrine tumors are able to synthesize and secrete PP. They occur mostly but not exclusively in the gastrointestinal tract, particularly in the rectum. The inactivation of the MEN 1 gene at 11q13 appears to be involved in the development of pancreatic but not of rectal PP-producing tumors.     

Preliminary experience with the Tsunami coronary stent: immediate and six-month clinical and angiographic results

The Tsunami is a new, balloon-expandable, stainless steel, tubular coronary stent whose design is based on a number of radial, diamond-shaped cells joined by double connectors. The aim of this two-centre, prospective, nonrandomized study was to examine the procedural, in-hospital, and long-term clinical and angiographic outcomes of patients undergoing angioplasty with the Tsunami stent. Sixty-one consecutive unselected patients were treated by means of the implantation of 74 Tsunami stents in 72 coronary lesions. Most of the patients (64%) had unstable angina or acute myocardial infarction. The baseline lesion morphology was complex in 76% of cases, and the mean lesion length was 14 +/- 6 mm. The procedural success rate was 98%. Mean percentage diameter of the stenosis decreased after the intervention from 79 +/- 12% to 10 +/- 6%. The in-hospital major adverse cardiac event rate was 3.3%. During the six-month follow-up, there was one cardiac death and nine subjects (14.5%) underwent target vessel revascularization. The six-month event-free survival rate was 80%. The angiographic restenosis rate was 17%: a focal or limited pattern (class I or II) was found in 43% of cases, whereas the remaining 57% had a proliferative morphology (class III or IV). In conclusion, this study indicates the good clinical and angiographic performance of the Tsunami coronary stent system in consecutive unselected patients.     

Nucleotide sequence,functional characterization and evolution of pFKN,a virulence plasmid in Pseudomonas syringae pathovar maculicola

Pseudomonas syringae pv. maculicola strain M6 (Psm M6) carries the avrRpm1 gene, encoding a type III effector, on a 40 kb plasmid, pFKN. We hypothesized that this plasmid might carry additional genes required for pathogenesis on plants. We report the sequence and features of pFKN. In addition to avrRpm1, pFKN carries an allele of another type III effector, termed avrPphE, and a gene of unknown function (ORF8), expression of which is induced in planta, suggesting a role in the plant-pathogen interaction. The region of pFKN carrying avrRpm1, avrPphE and ORF8 exhibits several features of pathogenicity islands (PAIs). Curing of pFKN (creating Psm M6C) caused a significant reduction in virulence on Arabidopsis leaves. However, complementation studies using Psm M6C demonstrated an obvious virulence function only for avrRpm1. pFKN can integrate and excise from the chromosome of Psm M6 at low frequency via homologous recombination between identical sequence segments located on the chromosome and on pFKN. These segments are part of two nearly identical transposons carrying avrPphE. The avrPphE transposon was also detected in other strains of P. s. pv. maculicola and in P. s. tomato strain DC3000. The avrPphE transposon was found inserted at different loci in different strains. The analysis of sequences surrounding the avrPphE transposon insertion site in the chromosome of Psm M6 indicates that pFKN integrates into a PAI that encodes type III effectors. The integration of pFKN into this chromosomal region may therefore be seen as an evolutionary process determining the formation of a new PAI in the chromosome of Psm M6.     

The evolution of polar fish hemoglobin: a phylogenetic analysis of the ancestral amino acid residues linked to the root effect

Originating from a benthic ancestor, the suborder Notothenioidei (the dominant fish fauna component of the Antarctic sea) underwent a remarkable radiation, which led notothenioids to fill several niches. The ecological importance of notothenioids in Antarctica and their biochemical adaptations have prompted great efforts to study their physiology and phylogeny, with special attention to the evolutionary adaptation of the oxygen-transport system. We herewith report the evolutionary history of alpha- and beta-globins under the assumption of the molecular clock hypothesis as a basis for reconstructing the phylogenetic relationships among species. These studies have been extended to fish species of other latitudes, including the Arctic region. The northern and southern polar oceans have very different characteristics; indeed, in many respects the Antarctic and Arctic ichthyofaunas are more dissimilar than similar. Our results show that the inferred phylogeny of Arctic and Antarctic globins is different. Taking advantage of the wealth of information collected on structure and function of hemoglobins, we have attempted to investigate the evolutionary history of an important physiological feature in fish, the Root effect. The results suggest that the amino acid residues reported to play a key role in the Root effect may be regarded as ancestor characters, but the lack of this effect in extant species can hardly be associated with the presence of synapomorphies.     

Role of PKC-delta activity in glutathione-depleted neuroblastoma cells

Protein kinases C (PKCs) are a family of isoenzymes sensitive to oxidative modifications and involved in the transduction signal pathways that regulate cell growth. As such, they can act as cellular sensors able to intercept intracellular redox changes and promote the primary adaptive cell response. In this study, we have demonstrated that PKC isoforms are specifically influenced by the amount of intracellular glutathione (GSH). The greatest GSH depletion is associated with a maximal reactive oxygen species (ROS) production and accompanied by an increase in the activity of the delta isoform and a concomitant inactivation of alpha. ROS generation induced early morphological changes in GSH-depleted neuroblastoma cells characterized, at the intracellular level, by the modulation of PKC-delta activity that was involved in the pathway leading to apoptosis. When cells were pretreated with rottlerin, their survival was improved by the ability of this compound to inhibit the activity of PKC-delta and to counteract ROS production. These results define a novel role of PKC-delta in the cell signaling pathway triggered by GSH loss normally associated with many neurodegenerative diseases and clinically employed in the treatment of neuroblastoma.     

Copper-induced oxidative damage on astrocytes: protective effect exerted by human high density lipoproteins

In the present study, we confirmed that copper ions induce oxidative damage in human astrocytes in culture, as demonstrated by the significant increase in the levels of hydroperoxides and in the fluorescence intensity of the fluorescent probe dichloro-dihydrofluorescein diacetate (H(2)DCFDA). The compositional changes were associated with a significant decrease in cell viability in astrocytes treated with 10 microM Cu(++) with respect to control cells. Astrocytes incubated with copper ions in the presence of high density lipoproteins (HDL) isolated from plasma of normolipemic subjects showed lower levels of hydroperoxides and a higher cell viability with respect to cells oxidized alone. Moreover, a significant decrease in the levels of hydroperoxides was observed in oxidized astrocytes treated with HDL. These results demonstrate that HDL exert a protective role against lipid peroxidation. The protective effect could be related to the ability of HDL to bind metal ions at the lipoprotein surface and/or to a stimulation of the efflux of lipid hydroperoxides from cell membranes as demonstrated in other cell types. Oxidative damage of astrocytes was induced at a copper concentration similar to that observed in cerebrospinal fluid (CSF) of patients affected by neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's diseases (PD). Lipoprotein particles similar for density and chemical composition to plasma HDL were recently isolated in human CSF, therefore, the protective role exerted by HDL against Cu(++)-induced oxidative damage of astrocytes could be of physiological relevance.