Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.     

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.     

Biogeographical distribution and ecological ranges of benthic cyanobacteria in East Antarctic lakes

For the first time, the cyanobacterial diversity from microbial mats in lakes of Eastern Antarctica was investigated using microscopic and molecular approaches. The present study assessed the biogeographical distribution of cyanobacteria in Antarctica. Five samples were taken from four lakes spanning a range of different ecological environments in Larsemann Hills, Vestfold Hills and Rauer Islands to evaluate the influence of lake characteristics on the cyanobacterial diversity. Seventeen morphospecies and 28 16S rRNA gene-based operational taxonomic units belonging to the Oscillatoriales, Nostocales and Chroococcales were identified. The internal transcribed spacer was evaluated to complement the 16S rRNA gene data and showed similar but more clear-cut tendencies. The molecular approach suggested that potential Antarctic endemic species, including a previously undiscovered diversity, are more abundant than has been estimated by morphological methods. Moreover, operational taxonomic units, also found outside Antarctica, were more widespread over the continent than potential endemics. The cyanobacterial diversity of the most saline lakes was found to differ from the others, and correlations between the sampling depth and the cyanobacterial communities can also be drawn. Comparison with database sequences illustrated the ubiquity of several cyanobacterial operational taxonomic units and their remarkable range of tolerance to harsh environmental conditions.     

In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis

Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.     

RcsF is an outer membrane lipoprotein involved in the RcsCDB phosphorelay signaling pathway in Escherichia coli

The RcsCDB signal transduction system is an atypical His-Asp phosphorelay conserved in gamma-proteobacteria. Besides the three proteins directly involved in the phosphorelay, two proteins modulate the activity of the system. One is RcsA, which can stimulate the activity of the response regulator RcsB independently of the phosphorelay to regulate a subset of RcsB targets. The other is RcsF, a putative outer membrane lipoprotein mediating the signaling to the sensor RcsC. How RcsF transduces the signal to RcsC is unknown. Although the molecular and physiological signals remain to be identified, the common feature among the reported Rcs-activating conditions is perturbation of the envelope. As an initial step to explore the RcsF-RcsC functional relationship, we demonstrate that RcsF is an outer membrane lipoprotein oriented towards the periplasm. We also report that a null mutation in surA, a gene required for correct folding of periplasmic proteins, activates the Rcs pathway through RcsF. In contrast, activation of this pathway by overproduction of the membrane chaperone-like protein DjlA does not require RcsF. Conversely, activation of the pathway by RcsF overproduction does not require DjlA either, indicating the existence of two independent signaling pathways toward RcsC.     

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.     

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.     

Amplified fragment length polymorphism confirms reciprocal monophyly in Chrysomya putoria and Chrysomya chloropyga: a correction of reported shared mtDNA haplotypes

Reinvestigation of mitochondrial haplotypes previously reported to be shared between the Afrotropical blowflies Chrysomya putoria Weidemann and Chrysomya chloropyga Weidemann (Diptera: Calliphoridae) revealed an error resulting from the misidentification of specimens. Preliminary amplified fragment length polymorphism (AFLP) analysis of the original and additional individuals again failed to find reciprocal monophyly, leading to a re-examination of the specimens for diagnostic male genitalic characters that were first described following the earlier study. Four of the original study specimens were found to have been misidentified, and definitive analysis of both mtDNA and AFLP genotypes using phylogenetic analysis and genetic assignment showed that each species was indeed reciprocally monophyletic. In addition to correcting the earlier error, this study illustrates how AFLP analysis can be used for efficient and effective specimen identification through both phylogenetic analysis and genetic assignment, and suggests that the latter method has special advantages for identification when no conspecific specimens are represented in the reference database.