Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin. Our results showed that LXA4 (1-20 ng/paw or 5 microg/kg i.p.) was effective in inhibiting carrageenan-induced paw edema from 30 min to 2 h. LXA4 (10 ng/paw) was also able to acutely inhibit PAF-, histamine-, PGE2- or bradykinin-induced paw edema, as well as the PAF-induced myeloperoxidase activity increase in the paws. Likewise, LXA4 (10 ng/cavity) also inhibited the pleural edema triggered by histamine (1h), and this response was not followed by leukocyte accumulation. Of note, the lipoxin receptor (ALX-r) antagonist Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, 200 ng/paw) significantly reverted the anti-edematogenic effect of ASA (300 mg/kg p.o.) against carrageenan, PAF, PGE2 and BK, without affecting the anti-edematogenic action caused by indomethacin (3 mg/kg i.p.) in the carrageenan-induced paw edema. Collectively, our results demonstrate for the first time that LXA4 displays an acute and rapid onset anti-edematogenic activity that does not discriminate among different pro-inflammatory stimuli, an effect that is most likely independent of its action on the leukocyte influx. Finally, the present study demonstrates that ATLs exert a very important role in the acute anti-edematogenic action of ASA.     

Structural basis for altering the stability of homologous RNAs from a mesophilic and a thermophilic bacterium

Tertiary RNA structures from thermophilic bacteria generally are more stable than their mesophilic homologs. To understand the structural basis of the increase in stability, we investigated equilibrium folding of the specificity domain (S-domain) of RNase P RNA from a mesophilic (Escherichia coli) and a thermophilic (Thermus thermophilus) bacterium. Equilibrium folding of both S-domains is described by a minimal, three-state folding scheme, U-to-I-to-N. In the I-to-N transition of the thermophilic S-domain, more structure forms and protections are stronger against T1 nuclease and hydroxyl radical reactions. Phylogenetic comparison in the context of the native structure reveals that among 39 nucleotide differences between these S-domains, 12 likely contribute to higher stability. These residues participate in extensive networks of hydrogen bonding, stacking, and metal ion coordination throughout the molecule. The thermophilic S-domain achieves higher stability by mutating strategic base pairs to G-C, decreasing surface accessibility of the native state, and increasing the amount of structure formation in the native folding transition. An E. coli S-domain mutant containing these 12 nt has the same stability and folding cooperativity as the T. thermophilus S-domain. E. coli S-domain mutants containing a subset of 4 or 6 nt have the same stability as the T. thermophilus S-domain but the same folding cooperativity as the E. coli S-domain. These results show that increasing stability can be accomplished by mutations within a local structure, but increasing folding cooperativity needs concerted changes among multiple structural units.     

Automatic clustering of orthologs and inparalogs shared by multiple proteomes

MOTIVATION: The complete sequencing of many genomes has made it possible to identify orthologous genes descending from a common ancestor. However, reconstruction of evolutionary history over long time periods faces many challenges due to gene duplications and losses. Identification of orthologous groups shared by multiple proteomes therefore becomes a clustering problem in which an optimal compromise between conflicting evidences needs to be found. RESULTS: Here we present a new proteome-scale analysis program called MultiParanoid that can automatically find orthology relationships between proteins in multiple proteomes. The software is an extension of the InParanoid program that identifies orthologs and inparalogs in pairwise proteome comparisons. MultiParanoid applies a clustering algorithm to merge multiple pairwise ortholog groups from InParanoid into multi-species ortholog groups. To avoid outparalogs in the same cluster, MultiParanoid only combines species that share the same last ancestor. To validate the clustering technique, we compared the results to a reference set obtained by manual phylogenetic analysis. We further compared the results to ortholog groups in KOGs and OrthoMCL, which revealed that MultiParanoid produces substantially fewer outparalogs than these resources. AVAILABILITY: MultiParanoid is a freely available standalone program that enables efficient orthology analysis much needed in the post-genomic era. A web-based service providing access to the original datasets, the resulting groups of orthologs, and the source code of the program can be found at http://multiparanoid.cgb.ki.se.     

X-ray structural studies of the fungal laccase from Cerrena maxima

Laccases are members of the blue multi-copper oxidase family. These enzymes oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and following the transfer of four electrons is reduced to two molecules of water. The X-ray structure of a laccase from Cerrena maxima has been elucidated at 1.9 A resolution using synchrotron data and the molecular replacement technique. The final refinement coefficients are Rcryst = 16.8% and Rfree = 23.0%, with root mean square deviations on bond lengths and bond angles of 0.015 A and 1.51 degrees , respectively. The type 1 copper centre has an isoleucine residue at the axial position and the "resting" state of the trinuclear centre comprises a single oxygen (OH) moiety asymmetrically disposed between the two type 3 copper ions and a water molecule attached to the type 2 ion. Several carbohydrate binding sites have been identified and the glycan chains appear to promote the formation of well-ordered crystals. Two tyrosine residues near the protein surface have been found in a nitrated state.     

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.     

Structure of the alpha-actinin-vinculin head domain complex determined by cryo-electron microscopy

The vinculin binding site on alpha-actinin was determined by cryo-electron microscopy of 2D arrays formed on phospholipid monolayers doped with a nickel chelating lipid. Chicken smooth muscle alpha-actinin was cocrystallized with the beta1-integrin cytoplasmic domain and a vinculin fragment containing residues 1-258 (vinculin(D1)). Vinculin(D1) was located at a single site on alpha-actinin with 60-70% occupancy. In these arrays, alpha-actinin lacks molecular 2-fold symmetry and the two ends of the molecule, which contain the calmodulin-like and actin binding domains, are held in distinctly different environments. The vinculin(D1) difference density has a shape very suggestive of the atomic structure. The atomic model of the complex juxtaposes the alpha-actinin binding site on vinculin(D1) with the N-terminal lobe of the calmodulin-like domain on alpha-actinin. The results show that the interaction between two species with weak affinity can be visualized in a membrane-like environment.     

Effect of NaCl and CaCl2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers

The influence of addition of NaCl or CaCl₂ (0.3 and 0.1 M, respectively) on the lateral diffusion coefficient (D_L) of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) was measured by the pulsed field gradient NMR technique. D_L of DOPC was unaffected, whereas the DOPG diffusion decreased with salt concentration. ²³Na NMR quadrupole splittings of DOPG between 20 and 60 °C and added NaCl between 0 and 15 wt% decreased only slightly with salt content, but increased with increasing temperature. Similar results were obtained for palmitoyloleoylphosphatidylglycerol, in which the palmitoyl chain order parameter increased slightly with salt. A model with free and “bound” ions was used to interpret the splitting data.With increasing salt content a decrease in the water permeability for DOPG was observed, but not for DOPC, as measured by water diffusion perpendicular to the oriented lipid bilayers.It was concluded that calcium and sodium ions interacted with the DOPG head-groups resulting in a decrease in the “free area” per lipid molecule due to a screening of the charged lipid head-groups. Thus, there was a closer packing of DOPG, leading to a decrease in D_L and water permeability. DOPC did not show any changes in the bilayer properties upon the addition of ions.     

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.Huntington disease (HD)⁴ is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt) protein above a threshold length of ∼36 (1). The deposition of polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark of HD, and IBs are found in human post-mortem samples, transgenic mouse brain, and cell-culture models (2). The accumulation of ubiquitinated proteins in IBs has implicated the ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson disease, and polyQ-mediated disorders (3).The UPS is a major pathway of intracellular protein degradation. After a series of three reactions, each catalyzed by a different set of enzymes, ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the amino group of lysine residues on substrate proteins. Several lysine residues within ubiquitin are sites for more ubiquitin additions. Once a protein accumulates four or more ubiquitins, it is efficiently targeted to the proteasome for degradation. The proteasome binds polyubiquitinated substrates and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before degrading substrate proteins through chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities (3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany the accumulation of UPS substrates in the brains of HD patients and transgenic mice and in cellular HD models (4). UPS substrates accumulate throughout the cell in polyQ models, even before IB formation (5, 6). This has added to the confusion over whether polyQ expansion leads to toxicity through direct impairment of proteasomal degradation. Proteasomes have been reported to cleave polyQ stretches efficiently (7), inefficiently (8), or essentially not at all (9). In vivo, polyQ-dependent degeneration occurs with no detectable proteasome inhibition (10, 11) or is tightly linked to it (12, 13). The inability of some studies to detect UPS impairment in HD models may be due to the limited sensitivity of conventional approaches to identify cell-to-cell variations in UPS function.The relationship between IB formation and UPS function has been difficult to determine. Protein turnover in cells with IBs is evidently reduced and accompanied by the accumulation of cellular proteins (14–16); HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment than those without IBs (5). Proteasome subunits and heat shock proteins colocalize with IBs, but it is unclear if this colocalization facilitates protein delivery or unfolding at the mouth of active proteasomes, or if it harms proteasome function by sequestering essential cellular machinery (18). Some IBs are relatively static (8, 25), but the proteins in others are dynamically exchanged with cytoplasmic and nuclear pools (19, 20).UPS function is critical to cellular homeostasis. Deletion of one of the two inducible polyubiquitin genes in mice leads to lower intracellular ubiquitin levels in germ cells and hypothalamic neurons. These same populations undergo cell-cycle arrest and hypothalamic neurodegeneration, respectively (22, 23). Cell lines expressing mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle arrest in G2/M (5). In neurons, UPS impairment may lead to cell death through an accumulation of signals for apoptosis, a decrease in NF-κB signaling, sensitization to other toxic stimuli, remodeling of synapses, retraction of neurites, or other unidentified mechanisms (24). The effect of UPS impairment depends on cell type and cell cycle, and the relationship between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a protein concentration-dependent manner (2). IB formation delays neuronal death, suggesting that IB formation helps neurons cope with toxic diffuse mutant htt. Whether the effect of IB formation on survival is mediated through UPS function has been difficult to determine. IB formation and neuronal death occur asynchronously in overlapping but distinct subsets of neurons that express mutant htt. The observation that IB formation is not required for UPS impairment also complicates population analysis (6, 26).To explore this problem, we applied single-cell analysis. We tracked single neurons over their entire lifetimes, gaining spatial and temporal resolution while simultaneously monitoring IB formation, UPS inhibition, and neuronal toxicity.