Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

This study shows that array comparative genomic hybridization (CGH) analysis is a useful tool for the molecular diagnosis of XY-GD as well as for the identification of potential candidate genes involved in male sexual development.

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Hepatitis C virus (HCV) isHepatitis C virus (HCV) is a major cause of posttransfusion and community-acquired hepatitis in the world (8,24,25). The majority of HCV-infected individuals develop chronic hepatitis that may progress to liver cirrhosis and hepatocellular carcinoma (15). Treatment options for chronic HCV infection are limited, and a vaccine to prevent HCV infection is not available (11,15,17).HCV has been classified in a separate genus (Hepacivirus) of the Flaviviridae family. The virion contains a positive-stranded RNA genome of approximately 9.6 kb in length (25). The genome consists of a highly conserved 5Ј noncoding region followed by a long open reading frame of 9,030 to 9,099 nucleotides that is translated into a single polyprotein of 3,010 to 3,030 amino acids. Processing of the polyprotein occurs by a combination of host and viral proteases. The HCV structural proteins comprise the putative nucleocapsid or core protein and the two envelope glycoproteins E1 and E2 (25). The E2 glycoprotein is thought to be responsible for initiating virus attachment due to its ability to bind to human cells (32).HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density lipoprotein receptor for uptake (2). In the absence of highly purified native infectious HCV particles as a tool for the study of virus-cell interaction, recombinant HCV glycoprotein E2, E1E2 liposomes (22), infectious HCV pseudotype particles expressing E1 and E2 (HCVpp) (4, 16), and HCV-like particles (HCV-LPs) (3,39,41) have been used to analyze virus-cell membrane interaction. Based on these experimental in vitro studies, CD81 (5, 32), dendritic cellspecific intercellular adhesion molecule 3 grabbing nonintegrin (33) and highly sulfated heparan sulfate (3) have been proposed to play a role in mediating E2 binding and/or HCV internalization.Recently, the scavenger receptor class B type I (SR-BI) has been proposed as a putative HCV receptor candidate (36). SR-BI, a high-density lipoprotein-binding molecule, plays a functional role in lipid metabolism and is highly expressed in hepatocytes and steroidgenic tissues (34). SR-BI is a 509-residue glycoprotein with a large extracellular loop (LEL) anchored to the plasma membrane at both the N and C termini by transmembrane domains with short extensions into the cytoplasm (21). SR-BI has been shown to play a role in mediating the binding of recombinant E2 to HepG2 hepatoma cells and the entry of recombinant HCVpp into Huh-7 hepatoma cells (5, 36). Due to the lack of convenient in vitro or in vivo models for the study of HCV infection, the

Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 Å. The enzyme is a 75-kDa diheme c -type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O 2 stably bound to the active site heme iron. Activation and cleavage of O 2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.

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The metal-reducing δ-proteobacterium Geobacter sulf urreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulf urreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS −2 as an electron donor. The observed K M was 38.5 ± 3.7 μM H 2 O 2 and v max was 0.78 ± 0.03 μmol of H 2 O 2 •min −1 •mg −1 , resulting in a turnover number k cat = 0.46 • s −1 . In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

The skin’s epidermis is a multilayered epithelial tissue and the first line of defense against mechanical stress. Its barrier function depends on an integrated assembly and reorganization of cell–matrix and cell–cell junctions in the basal layer and on different intercellular junctions in suprabasal layers. However, how mechanical stress is recognized and which adhesive and cytoskeletal components are involved are poorly understood. Here, we subjected keratinocytes to cyclic stress in the presence or absence of intercellular junctions. Both states not only recognized but also responded to strain by reorienting actin filaments perpendicular to the applied force. Using different keratinocyte mutant strains that altered the mechanical link of the actin cytoskeleton to either cell–matrix or cell–cell junctions, we show that not only focal adhesions but also adherens junctions function as mechanosensitive elements in response to cyclic strain. Loss of paxillin or talin impaired focal adhesion formation and only affected mechanosensitivity in the absence but not presence of intercellular junctions. Further analysis revealed the adherens junction protein α-catenin as a main mechanosensor, with greatest sensitivity conferred on binding to vinculin. Our data reveal a mechanosensitive transition from cell–matrix to cell–cell adhesions on formation of keratinocyte monolayers with vinculin and α-catenin as vital players.