Synthesis, characterization and X-ray structure of [Pd(SO4)(dppp)] · H2O, a catalyst for the CO-ethene copolymerization [dppp = 1,3-bis(diphenylphosphino)propane]

The title complex has been synthesized by first reacting dppp with Pd(AcO)₂ in acetone and then with NaHSO₄ in water. It has been characterized by IR, NMR and X-ray diffraction studies. The ³¹P NMR spectrum in DMSO shows a singlet at 16.62 ppm indicating that the two P atoms are equivalent and that the sulfate anion is weakly coordinating. The X-ray structure shows that the Pd atom is surrounded in an almost regular square planar environment by the two P atoms and by two O atoms of the sulfate anion and that the neutral complex is accompanied by a water molecule of crystallization. The Pd-P distances (2.217(1) and 2.233(1)) and the P-Pd-P angle (90.78(3)°) are close to those found in other complexes where the chelating diphosphine is the same. Also the Pd-O distances and the O-Pd-O bond angle are comparable to those of other relevant chelating ligands.In MeOH, the title complex, in combination with H₂SO₄, catalyses the CO-ethene copolymerization. The productivity reaches a maximum upon increasing the H₂SO₄/Pd ratio up to ca. 470 (7650 g of polyketone/g Pd h at 90 °C and 45 atm, CO/ethene 1/1). The viscosity of the polyketone passes through a maximum of 0.95 dL/g in m-cresol when the above ratio is ca. 100. It has been proposed that acid promotes the copolymerization process by destabilizing the β- and γ-chelates intermediates involved in chain growing process, thus favoring the insertion of the monomers. At relatively high acid concentration the lowering of productivity and viscosity suggests that the sulfate anion competes with the monomers for the coordination to the metal center.In H₂O-CH₃COOH as a solvent the productivity strongly depends on the H₂O/CH₃COOH ratio, as it passes through a maximum of 12 000 g polymer/g Pd h in the presence of ca. 60% of H₂O. The productivity is significantly lower than that found when the acetate and chloride analogues are used (27 000 g polyketone/g Pd · h). Thus, it is likely that the sulfate anion assists significantly the copolymerization process even though the concentration of CH₃COOH/CH₃COO⁻ is much preponderant.     

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate-derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.     

Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.     

Site-specific chlorophyll reference conditions for lakes in Northern and Western Europe

The Water Framework Directive (WFD) requires EU Member States to assess the “ecological status” of surface waters. As a component of ecological status, many European countries are developing a classification scheme for chlorophyll concentrations as a measure of phytoplankton biomass. The chlorophyll classification must be based on the degree of divergence of a water body from an appropriate baseline or ‘reference condition’. This article describes the development of a series of regression models for predicting reference chlorophyll concentrations on a site-specific basis. For model development, a large dataset of European lakes considered to be in reference condition, 466 lakes in total, was assembled. Data were included from 12 European countries, but lakes from Northern and Western Europe dominated and made up 92% of all reference lakes. Data have been collated on chlorophyll concentration, altitude, mean depth, alkalinity, humic type, surface area and geographical region. Regression models were developed for estimating site-specific reference chlorophyll concentrations from significant predictor ‘typology’ variables. Reference chlorophyll concentrations were found to vary along a number of environmental gradients. Concentrations increased with colour and alkalinity and decreased with lake depth and altitude. Forward selection was used to identify independent explanatory variables in regression models for predicting site-specific reference chlorophyll concentrations. Depth was selected as an explanatory variable in all models. Alkalinity was included in models for low colour and humic lakes and altitude was included in models for low colour and very humic lakes. Uncertainty in the models was quite high and arises from errors in the data used to develop the models (including natural temporal and spatial variability in data) and also from additional explanatory variables not considered in the models, particularly nutrient concentrations, retention time and grazing. Despite these uncertainties, site-specific reference conditions are still recommended in preference to type-specific reference conditions, as they use the individual characteristics of a site known to influence phytoplankton biomass, rather than adopt standards set to generally represent a large population of lakes of a particular type. For this reason, site-specific reference conditions should result in reduced error in ecological status classifications, particularly for lakes close to typology boundaries.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

Oxidosqualene cyclase from Saccharomyces cerevisiae,Trypanosoma cruzi,Pneumocystis carinii and Arabidopsis thaliana expressed in yeast: A model for the development of novel antiparasitic agents

A series of 25 compounds, some of which previously were described as inhibitors of human liver microsomal oxidosqualene cyclase (OSC), were tested as inhibitors of Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Arabidopsis thaliana OSCs expressed in an OSC-defective strain of S. cerevisiae. The screening identified three derivatives particularly promising for the development of novel anti-Trypanosoma agents and eight derivatives for the development of novel anti-Pneumocystis agents.     

Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases

Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.DEP-1⁴ (also known as CD148, HPTPη, and PTPRJ) is a class III receptor protein-tyrosine phosphatase, characterized by eight fibronectin type III repeats within the extracellular domain, a trans-membrane region, and a single cytosolic catalytic domain (1, 2). DEP-1 is expressed in all human hematopoietic cell lineages and was shown to negatively regulate T cell activation. In addition, several epithelial cell types display DEP-1 on their cell membranes (3). Homozygous DEP-1 mutant mice die before embryonic day 11.5, displaying severe defects in vascular organization (4). Interestingly, DEP-1 expression levels were found to augment with increased cell density (2), suggesting a role for this tyrosine phosphatase in sensing cell-cell contacts and in density-dependent growth inhibition (5). Moreover, accumulating evidence supports a prominent role for DEP-1 as a tumor suppressor as it negatively regulates cell proliferation and is poorly expressed in many cancer cell lines (6–10). The observed anti-proliferative effect may be accounted for by the ability of DEP-1 to down-regulate growth factor signaling through the dephosphorylation of various receptor tyrosine kinases, such as PDGFR, VEGFR2, and MET (11–13), resulting in quenching of the downstream RAS-MAPK pathway. However, given the complex pleiotropic functions of DEP-1, it is also possible that additional regulatory circuits mediated by yet unknown DEP-1 substrates may play a functional role in contact inhibition and control of cell proliferation.A variety of in vivo and in vitro approaches has led us to propose a number of DEP-1 substrates as mediators of its function. These include PDGFR, p120 catenin (CTND1), hepatocyte growth factor receptor, SRC kinase, VEGFR2, phosphatidylinositol 3-kinase regulatory subunit α (P85A), and RET receptor kinase (5, 11–16).Here we report a novel, unbiased strategy based on the screening of high density phosphopeptide arrays for their ability to bind phosphatase trapping mutants. A large portion of the phosphoproteome could be explored by this approach, thus unveiling a long list of potential substrates. A selected list of potentially relevant substrates has been obtained by applying a bioinformatics context filter. In this study we report the detailed characterization of one of these substrates, and we propose that DEP-1 modulates the RAS pathway by directly dephosphorylating Tyr-204 of ERK1/2. In addition, we show that the efficient removal of the phosphate group from Tyr-204 requires the integrity of a docking site on the ERK1/2 proteins.     

EAACI position paper on occupational rhinitis

The present document is the result of a consensus reached by a panel of experts from European and non-European countries on Occupational Rhinitis (OR), a disease of emerging relevance which has received little attention in comparison to occupational asthma. The document covers the main items of OR including epidemiology, diagnosis, management, socio-economic impact, preventive strategies and medicolegal issues. An operational definition and classification of OR tailored on that of occupational asthma, as well as a diagnostic algorithm based on steps allowing for different levels of diagnostic evidence are proposed. The needs for future research are pointed out. Key messages are issued for each item.