Crystal structure of Pseudomonas aeruginosa lipase in the open conformation - The prototype for family I.1 of bacterial lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 Angstrom resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two P-strands and one alpha-helix (alpha E) are not present. The absence of helix alpha E allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His251 is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme

Directed evolution of an enantioselective Bacillus subtilis lipase

Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-2-cyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural data to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations. This combined approach should finally enable us to devise more efficient strategies for the directed evolution of enantioselective enzymes

Connectionist perspectives on language learning, representation and processing.

The field of formal linguistics was founded on the premise that language is mentally represented as a deterministic symbolic grammar. While this approach has captured many important characteristics of the world\u27s languages, it has also led to a tendency to focus theoretical questions on the correct formalization of grammatical rules while also de-emphasizing the role of learning and statistics in language development and processing. In this review we present a different approach to language research that has emerged from the parallel distributed processing or \u27connectionist\u27 enterprise. In the connectionist framework, mental operations are studied by simulating learning and processing within networks of artificial neurons. With that in mind, we discuss recent progress in connectionist models of auditory word recognition, reading, morphology, and syntactic processing. We argue that connectionist models can capture many important characteristics of how language is learned, represented, and processed, as well as providing new insights about the source of these behavioral patterns. Just as importantly, the networks naturally capture irregular (non-rule-like) patterns that are common within languages, something that has been difficult to reconcile with rule-based accounts of language without positing separate mechanisms for rules and exceptions

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

The relationship between smokers' motivation to quit and intensity of tobacco control at the population level: a comparison of five European countries

Background: Smoking prevalence differs significantly across Europe. In addition, there are considerable differences in tobacco control activities across European countries. The relationship between prevalence and policy is under-researched. The present analysis examines the motivation to change smoking behaviour across 5 different European countries that differ considerably in their tobacco control activities. Methods: A population-based, representative survey of 1750 smokers, aged 16–59, from 5 different European countries (Germany, Greece, Poland, Sweden, UK) was used. Demographic variables, smoking status and the motivation to stop smoking were assessed. Motivation was assessed as, first, intending to quit (using the stages of change plus a modified stage for Precontemplation), and second, the desire to quit. Results: The majority of smokers want to stop smoking (73.5%), while only 35.0% want to stop definitely. Across countries, 10.2% definitely do not want to stop. Most of the smokers can be categorised in the Precontemplation stage (between 62.6% and 77.7% depending on the country), one of the stages of change categories. The relationship between the stages of change and the country under examination is statistically significant (chi-square = 43.466, p < 0.001). In countries with a high level of tobacco control, the proportion of people in Precontemplation is lower than in countries with low tobacco control activity. Conclusion: There are differences in the stages of change between the countries under examination. However, the categorisation of the countries into low, medium and high tobacco control activity used in this analysis does not explain these differences. Most smokers want to stop smoking, but a high proportion cannot indicate a time-frame when this is going to happen. Tobacco control efforts or other kinds of support might encourage these smokers to actually try to stop. Longitudinal studies at the population level are needed to assess, relate or monitor tobacco control activities and the intention to stop

Depleted 15N in hydrolysable-N of arctic soils and its implication for mycorrhizal fungi–plant interaction

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Biogeochemistry 97 (2009): 183-194, doi:10.1007/s10533-009-9365-1.Uptake of nitrogen (N) via root-mycorrhizal associations accounts for a significant portion of total N supply to many vascular plants. Using stable isotope ratios (δ15N) and the mass balance among N pools of plants, fungal tissues, and soils, a number of efforts have been made in recent years to quantify the flux of N from mycorrhizal fungi to host plants. Current estimates of this flux for arctic tundra ecosystems rely on the untested assumption that the δ15N of labile organic N taken up by the fungi is approximately the same as the δ15N of bulk soil. We report here hydrolysable amino acids are more depleted in 15N relative to hydrolysable ammonium and amino sugars in arctic tundra soils near Toolik Lake, Alaska, USA. We demonstrate, using a case study, that recognizing the depletion in 15N for hydrolysable amino acids (δ15N = -5.6 ‰ on average) would alter recent estimates of N flux between mycorrhizal fungi and host plants in an arctic tundra ecosystem.This study was funded by NSF-DEB-0423385and NSF-DEB 0444592. Additional support was provided by Arctic Long Term Ecological Research program, funded by National Science Foundation, Division of Environmental Biology

Human Primary Adipocytes Exhibit Immune Cell Function: Adipocytes Prime Inflammation Independent of Macrophages

BACKGROUND: Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT. METHODS AND RESULTS: Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%. CONCLUSION: Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes

DATABASE OF HOMOLOGY-DERIVED PROTEIN STRUCTURES AND THE STRUCTURAL MEANING OF SEQUENCE ALIGNMENT

The database of known protein three-dimensional structures can be significantly increased by the use of sequence homology, based on the following observations. (1) The database of known sequences, currently at more than 12,000 proteins, is two orders of magnitude larger than the database of known structures. (2) The currently most powerful method of predicting protein structures is model building by homology. (3) Structural homology can be inferred from the level of sequence similarity. (4) The threshold of sequence similarity sufficient for structural homology depends strongly on the length of the alignment. Here, we first quantify the relation between sequence similarity, structure similarity, and alignment length by an exhaustive survey of alignments between proteins of known structure and report a homology threshold curve as a function of alignment length. We then produce a database of homology-derived secondary structure of proteins (HSSP) by aligning to each protein of known structure all sequences deemed homologous on the basis of the threshold curve. For each known protein structure, the derived database contains the aligned sequences, secondary structure, sequence variability, and sequence profile. Tertiary structures of the aligned sequences are implied, but not modeled explicitly. The database effectively increases the number of known protein structures by a factor of five to more than 1800. The results may be useful in assessing the structural significance of matches in sequence database searches, in deriving preferences and patterns for structure prediction, in elucidating the structural role of conserved residues, and in modeling three-dimensional detail by homology