Pharmacological concentration of angiotensin-(1-7) activates NADPH oxidase after ischemia-reperfusion in rat heart through AT1 receptor stimulation

The cardiovascular role of angiotensin-(1-7), especially in the functional and metabolic alterations associated with ischemia-reperfusion (IR), is still not clearly defined. Our objective was to evaluate the cardiac effects of angiotensin-(1-7), the receptors involved, and their relationships with NADPH oxidase activation under non-ischemic conditions and, during an ischemia-reperfusion sequence. Isolated perfused rat hearts underwent 45 min of non-ischemic perfusion, or 30 min of global ischemia followed by 30 min of reperfusion. Angiotensin-(1-7) and/or AT1 receptor blocker losartan or angiotensin-(1-7) receptor antagonist (D-Ala7)-angiotensin-(1-7) were perfused. Our results showed that angiotensin-(1-7) was without effect at low concentrations (10(-10) to 10(-7) M). At a pharmacological concentration, 0.5 microM angiotensin-(1-7) induced vasoconstriction, which was antagonised by losartan. After ischemia, we noted a partial recovery of functional parameters, which was not modified by any of the treatments. The expression of AT1 receptor mRNA was increased by ischemia-reperfusion, except in (D-Ala7)-angiotensin-(1-7) treated hearts. Angiotensin-(1-7) further increased the AT1 expression. NADPH oxidase activity was enhanced in 0.5 microM angiotensin-(1-7)-treated hearts subjected to ischemia-reperfusion, this effect was totally reversed by losartan. This is the first time that it has been shown that, in the heart, angiotensin-(1-7) at pharmacological concentration activates NADPH oxidase, an enzyme thought to be involved in several angiotensin II effects.     

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. AVAILABILITY: MamMiBase is available at http://www.mammibase.lncc.br     

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.     

ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.     

Modification of photosynthetic regulation in tomato overexpressing glutathione peroxidase

To investigate the function of glutathione peroxidase (GPX) in plants, we produced transgenic tomato plants overexpressing an eukaryotic selenium-independent GPX (GPX5). We show here that total GPX activity was increased by 50% in transgenic plants, when compared to control plants transformed with the binary vector without the insert (PZP111). A preliminary two-dimensional electrophoretic protein analysis of the GPX overexpressing plants showed notably a decrease in the accumulation of proteins identified as rubisco small subunit 1 and fructose-1,6-bisphosphate aldolase, two proteins involved in photosynthesis. These observations, together with the fact that in standard culture conditions, GPX-overexpressing plants were not phenotypically distinct from control plants prompted us to challenge the plants with a chilling treatment that is known to affect photosynthesis activity. We found that upon chilling treatment with low light level, photosynthesis was not affected in GPX-overexpressing plants while it was in control plants, as revealed by chlorophyll fluorescence parameters and fructose-1,6-biphosphatase activity. These results suggest that overexpression of a selenium-independent GPX in tomato plants modifies specifically gene expression and leads to modifications of photosynthetic regulation processes.     

Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.