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.     

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.     

Spatial variation in antler investment of Apennine red deer

Heterogeneity in resource availability and quality can trigger spatial patterns in the expression of sexually selected traits such as body mass and weaponry. While relationships between habitat features and phenotypic quality are well established at broad geographical scales, information is poor on spatial patterns at finer, intrapopulation scales. We analyzed biometric data collected on 1965 red deer Cervus elaphus males over 20 years from a nonmigratory population living on two sides of a mountainous ridge, with substantial differences in land cover and habitat quality but similar climate and population density. We investigate spatial patterns in (i) body mass, (ii) antler mass, and (iii) antler investment. We also tested for site‐ and age‐specific patterns in allometric relationship between body mass and antler mass. Statistically significant fine‐scale spatial variations in body mass, antler mass, and, to a lesser extent, antler allocation matched spatial differences in land cover. All three traits were greater in the northern slope, characterized by higher habitat heterogeneity and greater availability of open habitats, than in the southern slope. Moreover, the allometric relationship between body mass and antler mass differed among age‐classes, in a pattern that was consistent between the two mountain slopes. Our results support the occurrence of spatial patterns in the expression of individual attributes also at a fine, intrapopulation scale. Our findings emphasize the role of environmental heterogeneity in shaping spatial variations of key life‐history traits, with potential consequences for reproductive success.     

Cytokines and lymphocyte proliferation in patients with different clinical forms of chromoblastomycosis

Chromoblastomycosis is a chronic, often debilitating, suppurative, granulomatus mycosis of the skin and subcutaneous tissues beginning after inoculation trauma. It occurs world-wide, but is more frequently observed in tropical countries such as Brazil. The disease is usually insidious, and the lesions increase slowly but progressively, not responding to the usual treatments and quite often reappearing. The host defense mechanism in chromoblastomycosis has not been extensively investigated. Some studies have focused on fungus-host interaction, showing a predominantly cellular immune response, with the activation of macrophages involved in fungus phagocytosis. Although phagocytosis did occur, death of fungal cells was rarely observed. The ability of Fonsecaea pedrosoi to produce secreted or cell wall-associated melanin-like components, protects against destruction by host immune cells in vitro. Until now, the T cell immune response in chromoblastomycosis is undefined. In the present work, it was shown that, in patients with the severe form of the disease, predominant production of IL-10 cytokine, low levels of IFN-gamma and inefficient T cell proliferation were induced. In contrast, in patients with a mild form of the disease, predominant production of IFN-gamma cytokine, low levels of IL-10 and efficient T cell proliferation were observed.     

A critical reassessment of penetratin translocation across lipid membranes

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.     

Sphingolipid uptake by cultured cells: complex aggregates of cell sphingolipids with serum proteins and lipoproteins are rapidly catabolized

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-(3)H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.     

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.     

Chemical synthesis of mouse cripto CFC variants

We report for the first time the chemical synthesis of refolded CFC domain of mouse Cripto (mCFC) and of two variants bearing mutations on residues W107 and H104 involved in Alk4 binding. The domains undergo spontaneous and quantitative refolding in about 4 h, yet with very different kinetics. Disulfide linkages have been assessed by enzyme digestion and mass spectrometry analysis of resulting fragments, and the first experimental studies on structural organization have been conducted by circular dichroism spectroscopy under different pH conditions. Upon refolding, the domains considerably change their conformations, although they do not assume canonical structures, and become highly resistant to enzyme degradation. A comparative study of receptor binding shows that the CFC domain can bind Alk4 and confirms the importance of W107 and H104 for receptor recognition.     

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.