Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase

Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.     

Bronchiolitis obliterans with organizing pneumonia. Retrospective study of 19 cases

We report a series of 19 cases of bronchiolitis obliterans with organized pneumonitis (BOOP) observed in two pneumology units at the Strasbourg University Hospital between July 1987 and June 1997. Mean patient age was 60 years. Clinical features included dry cough, exercise-induced dyspnea, and a flu-like syndrome in three-quarters of the cases. Standard chest x-ray showed a diffuse non-systemized alveolar syndrome with a air bronchogram in 18 cases and an interstitial syndrome in one-third of the cases. computed tomography of the thorax visualized bronchial dilatations by traction of the alveolar syndrome in one-third of the cases. Pulmonary function tests showed moderate restriction. Lymphocytes predominated in bronchio-alveolar lavage fluid. Pathology examination of surgical lung specimens (5 cases), transbronchial biopsies (5 cases) and scan-guided transparietal punctures (4 cases) provided the diagnosis. In 5 cases the diagnosis was based on the radiological and clinical presentation and favorable course on corticosteroid therapy. Recurrence was observed at corticosteroid withdrawal or dose reduction in 7 cases. In this series, bronchiolitis obliterans with organized pneumonitis was probably secondary to rheumatoid arthritis (1 case), breast radiotherapy (3 cases), and drugs (amiodarone: 1 case: sotalol: 2 cases: betaxolol: 1 case). An association with betaxolol has not been previously reported in the literature.     

A process model of shiftwork and health.

The authors developed and tested a process model of adaptation to shiftwork, which hypothesizes that various individual and situational variables influence the development of sleep and social and domestic disturbances. Both types of disturbances trigger various types of coping behavior, leading to several proximal outcomes. The end result is the development of chronic health problems in the form of digestive and cardiovascular symptoms. The model was tested with survey data collected from 2 samples of nurses (N?=?1,532) in the United Kingdom and was cross-validated against a 3rd sample of industrial workers (N?=?370). Results indicate support for the model across the 3 samples, although some sample-specific and subgroup effects were found. Results have direct implications for the development of shiftwork theory and interventions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Identification of a microscopically selected microorganism in milk samples

Identification of unwanted microbial contaminants microscopically observed in food products is challenging due to their low abundance in a complex matrix, quite often containing other microorganisms. Therefore, a selective identification method was developed using laser capture microdissection in combination with direct-captured cell PCR. This procedure was validated with Geobacillus stearothermophilus and further used to identify microbial contaminants present in some industrial milk samples. The microscopically observed contaminants were identified as mainly Methylobacterium species.     

Electrochemical oxidation of 1,4‐dioxane at boron‐doped diamond electrode

BACKGROUND: The electrochemical oxidation of 1,4‐dioxane at a boron doped diamond (BDD) surface on a niobium substrate anode was studied because (i) 1,4 dioxane is a resistant contaminant in waste‐waters and ground‐waters which needs to be removed/oxidized and (ii) most of the currently applied techniques for removal/oxidation require chemicals. RESULTS: Results show that in the potential region supporting electrolyte stability 1,4‐dioxane can be oxidized directly. Adhesive products, which cause electrode fouling, are also formed during oxidation in this potential region. The BDD anode can be restored to its initial activity by simple anodic treatment in the potential region of electrolyte decomposition. In this region, oxidation reactions leading to complete oxidation of 1,4‐dioxane, can take place due to electro‐generated hydroxyl radicals. Therefore, dioxane can only be effectively oxidized at these potentials. The effect of current density on the oxidation of 1,4‐dioxane has been investigated. The experimental results have also been compared with a theoretical chemical oxygen demand (COD)–instantaneous current efficiency (ICE) model. At a current density above 32 mA cm^?2, the oxidation process is completely controlled by mass transfer and no intermediates are formed. 92% of the COD can be removed with a total consumption of 7 Ah L^?1. CONCLUSIONS: Results show that dioxane can be effectively and completely oxidized at a BDD anode. Copyright © 2010 Society of Chemical Industry     

Dissolved Silicon and Its Origin in Belgian Beers—A Multivariate Analysis

Beer is a quintessential part of Belgian heritage. We performed a detailed analysis of factors controlling Si content in Belgian beers as a case study to coincide with the 2011 IBiS meeting in Antwerp (Belgium). Beer is one of the richest dietary sources of Si. Three decades of research have yielded evidence of a role for Si in human physiology: it plays an essential role in bone mineral density and reduces the biological availability of aluminium. We analysed 119 Belgian beers: highest dissolved Si concentrations were found in high fermentation, traditionally brewed ales. Concentrations ranged between 214 and 2,071 μmol L^???1. This is probably due to the complexity and length of the brewing procedure: longer, more complicated processing and presence of brewing sediment in the bottle allows more Si to dissolve out of the base products like hop, barley or even rice. As a side effect of fermentation, alcohol content was related to Si content.     

Rapid and easy development of versatile tools to study protein/ligand interactions

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.     

Towards a better understanding of the pectin structure–function relationship in broccoli during processing: Part II — Analyses with anti-pectin antibodies

To investigate the structure–function relationship of pectin during (pre)processing, broccoli samples (Brassica oleracea L. cultivar italica) were subjected to one of the following pretreatments: (i) low-temperature blanching (LTB), (ii) LTB in combination with Ca²⁺ infusion, (iii) high-pressure pretreatment (HP), (iv) HP in combination with Ca²⁺ infusion, or (v) no pretreatment (control sample), whether or not in combination with a thermal treatment of 15 min at 90 °C. Anti-homogalacturonan antibodies were used to perform in situ (microscopy) and ex situ (immuno-dot assays) analyses on broccoli pectin which resulted in information concerning the localisation of defined pectic domains in broccoli cell walls and pectin's structure. Water-soluble pectin appears to contain unbranched, high-esterified pectin and some pectic polymers with abundant side chains that are less esterified. Ionically cross-linked pectin, on the other hand, contains low-esterified pectin with either highly branched or unbranched domains. The in situ visualisation of pectin in broccoli suggested that de-esterification of pectin by PME during LTB as well as during HP mainly takes place in the tricellular junctions of adjacent cells in broccoli tissue. Ca²⁺-cross-linked pectin could be found in cell walls lining intercellular spaces and was particularly abundant at the corners of intercellular spaces, indicating its important role in cell–cell adhesion. Both LTB and HP created pectin–Ca²⁺-cross-links in parts of the cell wall where these cross-links were originally absent. The influence of thermal processing and the effect of pressurisation on the pectic components in the cell wall could also be visualised using the antibodies.