Proteolysis by the ubiquitin/proteasome pathway controls the intracellular levels of a number of proteins that regulate cell proliferation and cell cycle progression. To determine whether this pathway of protein turnover was also linked to apoptosis, we treated Rat-1 and PC12 cells with specific proteasome inhibitors. The peptide aldehydes PSI and MG115, which specifically inhibit the chymotrypsin-like activity of the proteasome, induced apoptosis of both cell types. In contrast, apoptosis was not induced by inhibitors of lysosomal proteases or by an alcohol analog of PSI. The tumor suppressor p53 rapidly accumulated in cells treated with proteasome inhibitors, as did the p53-inducible gene products p21 and Mdm-2. In addition, apoptosis induced by proteasome inhibitors was inhibited by expression of dominant-negative p53, whereas overexpression of wild-type p53 was sufficient to induce apoptosis of Rat-1 cells in transient transfection assays. Although other molecules may also be involved, these results suggest that stabilization and accumulation of p53 plays a key role in apoptosis induced by proteasome inhibitors.

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specific probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Neuronal apoptosis is critical for normal development of the mammalian nervous system and also contributes to the pathogenesis of ischemic and degenerative diseases of the brain. Apoptosis of neurons is tightly regulated by extrinsic signals including growth factors and neuronal activity, but the intracellular mechanisms by which these signals promote neuronal survival are incompletely understood. We report that the transcription factor NFAT3 plays a critical role in mediating survival of granule neurons of the developing cerebellum. NFAT3 accumulated in the nucleus of primary granule neurons under survival conditions of serum growth factors and neuronal activity that was elicited by depolarization with high K ؉ . In contrast, deprivation of serum and K ؉ , which leads to neuronal apoptosis, triggered NFAT3 nuclear export. Treatment of granule neurons with Li ؉ , an inhibitor of the NFAT export kinase GSK3, prevented the nuclear export of NFAT3 and increased granule cell survival even under pro-apoptotic conditions. Thus, the nuclear localization of NFAT3 correlated tightly with granule neuron survival. Consistent with a pro-survival function for NFAT3, genetic knockdown of NFAT3 by RNA interference in primary granule neurons led to increased apoptosis even in neurons cultured under survival conditions. Conversely, expression of a constitutively active form of NFAT protected neurons against apoptosis induced by serum withdrawal and low K ؉ . Taken together, these results reveal an essential function for NFAT3-mediated transcription in neuronal survival that may play important roles in the developing and mature brain.

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor B (NF-B) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.A general consequence of infectious diseases that cause hemolysis, internal hemorrhage, or extensive cell damage is the release of hemeproteins. Upon oxidation, hemeproteins release heme, a potentially harmful molecule (1). Heme-binding plasma proteins, such as hemopexin or albumin, remove the intravascular free heme, subsequently degraded by heme oxygenase-1 (HO-1), generating equimolar amounts of biliverdin, carbon monoxide, and free iron (2, 3). HO-1-deficient mice (Hmox Ϫ/Ϫ ) have high plasma concentrations of heme and show increased susceptibility to LPS-induced lethality, associated with inflammation and oxidative damage (4). Accumulation of large amounts of heme might overwhelm the capacity of heme scavengers and degrading system, thus causing oxidative stress and inflammation (5, 6). In fact, recent studies suggest that heme, in combination with ROS 3 and inflammatory mediators, increase blood brain barrier leakage and hepatocyte necrosis in models of malarial infection (7,8).Hemolysis or hemoglobinemia are associated with increased mortality in septic patients (9, 10). Hemoglobin increases the secretion of TNF triggered by LPS, whereas globin has an inhibitory effect (11), suggesting that heme is responsible for the cytokine amplification. Heme has several pro-inflammatory activities, including leukocyte activation and migration, upregulation of adhesion molecules, ROS production, and induction of cytokine expression (12-14). Recently, we have shown that heme is able to activate Toll-like receptor 4 (TLR4) inducing TNF on macrophages and dendritic cells (DC) (15).Mammalian pattern recognition receptors (PRRs) recognize conserved microbial molecules from all classes of microorganisms (16,17). The activation of these receptors elicits selective intracellular signaling cascades that result in the production of cytokines, chemokin...

SummaryAs Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA 2 inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE 2 and PGI 2 . Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from noninfected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10 4 cfu of PA103 exhibited a marked influx of inflammatory cells and PGE 2 release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.