The role of CXCR2 activity in the contact hypersensitivity response in mice

The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.     

Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes

Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) have been implicated in the regulation of signal transduction. However, the cellular mechanisms that link Nox activation with plasma membrane receptor signaling remain poorly defined. We have found that Nox2-derived ROS influence the formation of an active interleukin-1 (IL-1) receptor complex in the endosomal compartment by directing the H2O2-dependent binding of TRAF6 to the IL-1R1/MyD88 complex. Clearance of both superoxide and H2O2 from within the endosomal compartment significantly abrogated IL-1beta-dependent IKK and NF-kappaB activation. MyD88-dependent endocytosis of IL-1R1 following IL-1beta binding was required for the redox-dependent formation of an active endosomal receptor complex competent for IKK and NF-kappaB activation. Small interfering RNAs to either MyD88 or Rac1 inhibited IL-1beta induction of endosomal superoxide and NF-kappaB activation. However, MyD88 and Rac1 appear to be recruited independently to IL-1R1 following ligand stimulation. In this context, MyD88 binding was required for inducing endocytosis of IL-1R1 following ligand binding, while Rac1 facilitated the recruitment of Nox2 into the endosomal compartment and subsequent redox-dependent recruitment of TRAF6 to the MyD88/IL-1R1 complex. The identification of Nox-active endosomes helps explain how subcellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.     

Carotenoid-bacteriochlorophyll energy transfer in LH2 complexes studied with 10-fs time resolution

In this report, we present a study of carotenoid-bacteriochlorophyll energy transfer processes in two peripheral light-harvesting complexes (known as LH2) from purple bacteria. We use transient absorption spectroscopy with approximately 10 fs temporal resolution, which is necessary to observe the very fast energy relaxation processes. By comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it has been possible to directly evaluate their energy transfer efficiency to the bacteriochlorophylls. In the case of okenone in the LH2 complex from Chromatium purpuratum, we obtained an energy transfer efficiency of etaET2=63+/-2.5% from the optically active excited state (S2) and etaET1=61+/-2% from the optically dark state (S1); for rhodopin glucoside contained in the LH2 complex from Rhodopseudomonas acidophila these values become etaET2=49.5+/-3.5% and etaET1=5.1+/-1%. The measurements also enabled us to observe vibrational energy relaxation in the carotenoids' S1 state and real-time collective vibrational coherence initiated by the ultrashort pump pulses. Our results are important for understanding the dynamics of early events of photosynthesis and relating it to the structural arrangement of the chromophores.     

A possible role of exon-shuffling in the evolution of signal peptides of human proteins

It was recently shown that there is a predominance of phase 1 introns near the cleavage site of signal peptides encoded by human genes. It was suggested that this biased distribution was due to intron insertion at AGmid R:G proto-splice sites. However, we found that there is no disproportional excess of AGmid R:G that would support insertion at proto-splice sites. In fact, all nGmid R:G sites are enriched in the vicinity of the cleavage site. Additional analyses support an alternative scenario in which exon-shuffling is largely responsible for such excess of phase 1 introns.     

Analysis of detergent-resistant membranes associated with apical and basolateral GPI-anchored proteins in polarized epithelial cells

Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.