Reduction of antigen-induced respiratory abnormalities and airway inflammation in sensitized guinea pigs by a superoxide dismutase mimetic

Reactive oxygen species have been implicated in the pathogenesis of asthma and, in atopic asthmatics, endogenous superoxide dismutase (SOD) enzyme levels are known to decrease. This suggests that replacing a failed endogenous SOD enzyme system with a mimetic of the endogenous enzyme would be beneficial and protective. In this study we demonstrate that removal of superoxide by the SOD mimetic (SODm) M40403 reduces the respiratory and histopathological lung abnormalities due to ovalbumin (OA) aerosol in a model of allergic asthma-like reaction in sensitized guinea pigs. Both respiratory abnormalities and bronchoconstriction in response to OA challenge are nearly absent in na?ve animals, while they sharply became severe in sensitized animals. In addition, OA aerosol induced a reduction of MnSOD activity which was paralleled with bronchiolar lumen reduction, pulmonary air space hyperinflation, mast cell degranulation, eosinophil infiltration, bronchial epithelial cell apoptosis, increase in myeloperoxidase activity, malonyldialdehyde production and 8-hydroxy-2'-deoxyguanosine formation in the lung tissue, as well as elevation of PGD2 in the bronchoalveolar lavage fluid. Treatment with M40403 restored the levels of MnSOD activity and significantly reduced all the above parameters. In summary, our findings support the potential therapeutic use of SOD mimetics in asthma and anaphylactic reactions and account for a critical role for superoxide in acute allergic asthma-like reaction in actively sensitized guinea pig.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.     

Molecular phylogeny of songbirds (Aves: Passeriformes) and the relative utility of common nuclear marker loci

While the monophyly of the largest avian order Passeriformes as well as its suborders suboscines (Tyranni) and oscines (Passeri) is well established, lower phylogenetic relationships of this fast radiated taxon have been a continuous matter of debate, especially within the suborder oscines. Many studies analyzing phylogenetic relationships of the Passeriformes using molecular markers have been published, which led to a better resolved phylogeny. Conflicting hypotheses and still remaining uncertainties, especially within the Passerida, have repeatedly stimulated further research with additional new markers. In the present study we used a combination of established molecular markers (RAG‐1, RAG‐2, c‐myc) and the recently introduced ZENK. We accomplished phylogenetic analyses using maximum parsimony, maximum likelihood and Bayesian inference, both separately for all genes and simultaneously. To assess the phylogenetic utility of the different genes in avian systematics we analyzed the influence of each data partition on the phylogenetic tree yielded by the combined approach using partitioned Bremer support. Compared with the other single gene analyses, the ZENK trees exhibited by far the best resolution and showed the lowest amount of homoplasy. Our data indicate that this gene is—at least in passerines—suitable for inference of even old taxonomic splits. Our combined analysis yields well‐supported phylogenetic hypotheses for passerine phylogeny and apart from corroborating recently proposed hypotheses on phylogenetic relationships in the Passeriformes we provide evidence for some new hypotheses. The subdivision of the Passerida into three superfamilies, Sylvioidea, Passeroidea and Muscicapoidea, the first as sister to the two latter groups is strongly supported. We found evidence for a split between Paridae and the remaining Sylvioidea. © The Willi Hennig Society 2007.     

GBF1- and ACBD3-Independent Recruitment of PI4KIIIβ to Replication Sites by Rhinovirus 3A Proteins

PI4KIIIβ recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIIIβ to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIβ to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIIIβ recruitment occurred independently of both GBF1 and ACBD3.     

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Lyme disease is the most common tick‐borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface‐exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE‐positive patients by mapping the protein as overlapping peptides and subsequent in‐depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE‐positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein‐Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.     

Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.