Motivated proteins: a web application for studying small three-dimensional protein motifs

<b>BACKGROUND:</b> Small loop-shaped motifs are common constituents of the three-dimensional structure of proteins. Typically they comprise between three and seven amino acid residues, and are defined by a combination of dihedral angles and hydrogen bonding partners. The most abundant of these are alphabeta-motifs, asx-motifs, asx-turns, beta-bulges, beta-bulge loops, beta-turns, nests, niches, Schellmann loops, ST-motifs, ST-staples and ST-turns.We have constructed a database of such motifs from a range of high-quality protein structures and built a web application as a visual interface to this. <b>DESCRIPTION:</b> The web application, Motivated Proteins, provides access to these 12 motifs (with 48 sub-categories) in a database of over 400 representative proteins. Queries can be made for specific categories or sub-categories of motif, motifs in the vicinity of ligands, motifs which include part of an enzyme active site, overlapping motifs, or motifs which include a particular amino acid sequence. Individual proteins can be specified, or, where appropriate, motifs for all proteins listed. The results of queries are presented in textual form as an (X)HTML table, and may be saved as parsable plain text or XML. Motifs can be viewed and manipulated either individually or in the context of the protein in the Jmol applet structural viewer. Cartoons of the motifs imposed on a linear representation of protein secondary structure are also provided. Summary information for the motifs is available, as are histograms of amino acid distribution, and graphs of dihedral angles at individual positions in the motifs. <b>CONCLUSION:</b> Motivated Proteins is a publicly and freely accessible web application that enables protein scientists to study small three-dimensional motifs without requiring knowledge of either Structured Query Language or the underlying database schem

Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

Examining the effects of sodium ions on the binding of antagonists to dopamine D2 and D3 receptors

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo

CD5-positive marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) of the lung

CD5-positive marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) of the lung is very rare. An 82-year-old Japanese woman was found to have an abnormal lung shadow on chest X-ray photography, and was admitted to our hospital. Imaging modalities including X-ray photography, computed tomography, and magnetic resonance imaging showed a small (2 × 1 × 1 cm) opacity of the right upper lobe. Transbronchial lung biopsy was performed. It showed severe proliferation of small lymphocytes. The small lymphocytes were centrocytes-like, and minor plasma cell differentiation was recognized. Lymphoepithelial lesions were scattered. Immunohistochemically, the tumor cells were positive for CD5, CD20, CD43, CD45, CD79α, bcl-2, and κ-chain, but negative for CD2, CD3, CD10, CD21, CD23, CD35, CD45RO, CD56, IgA, IgG, IgM, IgD, λ-chain, TdT, and cyclin D1. The Ki-67 labeling was 10%. CD3-positive and CD45RO-positive inflammatory T-cells were scattered in small amount. The pathological diagnosis was CD5-positive marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) of the lung. The patient was treated with chemotherapy (CHOP: cyclophosphamide, hydroxydaunorbicin, vincristine, and predonisone), and the lung tumor disappeared. The patient is now free of the lymphoma 10 years after the first manifestation

Screening and brief interventions for hazardous and harmful alcohol use in primary care: a cluster randomised controlled trial protocol

A large number of randomised controlled trials in health settings have consistently reported positive effects of brief intervention in terms of reductions in alcohol use. However,although alcohol misuse is common amongst offenders, there is limited evidence of alcohol brief interventions in the criminal justice field. This factorial pragmatic cluster randomised controlledtrial with Offender Managers (OMs) as the unit of randomisation will evaluate the effectiveness and cost-effectiveness of different models of screening to identify hazardous and harmful drinkers in probation and different intensities of brief intervention to reduce excessive drinking in probation clients. Ninety-six OMs from 9 probation areas across 3 English regions (the NorthEast Region (n = 4) and London and the South East Regions (n = 5)) will be recruited. OMs will berandomly allocated to one of three intervention conditions: a client information leaflet control condition (n = 32 OMs); 5-minute simple structured advice (n = 32 OMs) and 20-minute brieflifestyle counselling delivered by an Alcohol Health Worker (n = 32 OMs). Randomisation will be stratified by probation area. To test the relative effectiveness of different screening methods all OMs will be randomised to either the Modified Single Item Screening Questionnaire (M-SASQ) orthe Fast Alcohol Screening Test (FAST). There will be a minimum of 480 clients recruited into the trial. There will be an intention to treat analysis of study outcomes at 6 and 12 months postintervention. Analysis will include client measures (screening result, weekly alcohol consumption,alcohol-related problems, re-offending, public service use and quality of life) and implementation measures from OMs (the extent of screening and brief intervention beyond the minimum recruitment threshold will provide data on acceptability and feasibility of different models of brief intervention). We will also examine the practitioner and organisational factors associated with successful implementation.The trial will evaluate the impact of screening and brief alcohol intervention in routine probation work and therefore its findings will be highly relevant to probation teams and thus the criminal justice system in the UK

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

BACKGROUND: G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration. METHODOLOGY/PRINCIPAL FINDINGS: First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo. CONCLUSIONS/SIGNIFICANCE: These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors

Congenital Hypogonadotropic Hypogonadism Due to GNRH Receptor Mutations in Three Brothers Reveal Sites Affecting Conformation and Coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gαq/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote

Erosion consequences on beach functions along the Maresme coast (NW Mediterranean, Spain)

A methodology to analyse the influence of erosion on beach functions at a regional scale is presented. The method considers erosion hazards at different timescales and assesses consequences by evaluating impacts on recreation and protection functions. To provide useful information to decision makers for managing these functions, hazard and consequences are integrated at the municipal level within a risk matrix. This methodology is applied at the Maresme, a 45-km sandy coast situated northward of Barcelona, which supports a strong urban and infrastructure development as well as an intensive beach recreational use. Obtained results indicate differentiated erosion implications along the region, depending on the management target considered. Thus, southern municipalities are more prone to erosion affecting the protection function of the beach and leisure use by the local population, whereas erosion will have a greater effect on foreign tourism in the northern municipalities. These results highlight the necessity to employ an articulated erosion risk assessment focusing on specific targets depending on the site in question. This methodology can help coastal managers to adopt tailored measures to manage erosion impacts towards specific goals, in a more efficient and sustainable manner

Optimization of lipase production by solid-state fermentation of olive pomace: from flask to laboratory-scale packed-bed bioreactor

Lipases are versatile catalysts with many applications and can be produced by solid-state fermentation (SSF) using agro-industrial wastes. The aim of this work was to maximize the production of Aspergillus ibericus lipase under SSF of olive pomace (OP) and wheat bran (WB), evaluating the effect on lipase production of C/N ratio, lipids, phenols, content of sugars of substrates and nitrogen source addition. Moreover, the implementation of the SSF process in a packed-bed bioreactor and the improvement of lipase extraction conditions were assessed. Low C/N ratios and high content of lipids led to maximum lipase production. Optimum SSF conditions were achieved with a C/N mass ratio of 25.2 and 10.2% (w/w) lipids in substrate, by the mixture of OP:WB (1:1) and supplemented with 1.33% (w/w) (NH4)2SO4. Studies in a packed-bed bioreactor showed that the lower aeration rates tested prevented substrate dehydration, improving lipase production. In this work, the important role of Triton X-100 on lipase extraction from the fermented solid substrate has been shown. A final lipase activity of 223 ± 5 U g1 (dry basis) was obtained after 7 days of fermentation.Felisbela Oliveira acknowledges the financial support from Fundação para a Ciência e Tecnologia (FCT) of Portugal through grant SFRH/BD/87953/2012. José Manuel Salgado was supported by grant CEB/N2020–INV/01/2016 from Project ‘‘BIOTECNORTE-Underpinning Biotechnology to foster the north of Portugal bioeconomy’’ (NORTE-01-0145-FEDER-000004). Luı ´s Abrunhosa was supported by grant UMINHO/BPD/51/2015 from project UID/BIO/04469/2013 financed by FCT/MEC (OE). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) and BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. Noelia Pérez-Rodríguez acknowledges the financial support of FPU fellowship from the Spanish Ministry of Education, Culture and Sports. The authors thank the Spanish Ministry of Economy and Competitiveness for the financial support of this work (Project CTQ2015-71436-C2-1-R), which has partial financial support from the FEDER funds of the European Union.info:eu-repo/semantics/publishedVersio

Marine Invasion in the Mediterranean Sea: The Role of Abiotic Factors When There Is No Biological Resistance

The tropical red alga Womersleyella setacea (Rhodomelaceae, Rhodophyta) is causing increasing concern in the Mediterranean Sea because of its invasive behavior. After its introduction it has colonized most Mediterranean areas, but the mechanism underlying its acclimatization and invasion process remains unknown. To understand this process, we decided i) to assess in situ the seasonal biomass and phenological patterns of populations inhabiting the Mediterranean Sea in relation to the main environmental factors, and ii) to experimentally determine if the tolerance of W. setacea to different light and temperature conditions can explain its colonization success, as well as its bathymetric distribution range. The bathymetric distribution, biomass, and phenology of W. setacea were studied at two localities, and related to irradiance and temperature values recorded in situ. Laboratory experiments were set up to study survival, growth and reproduction under contrasting light and temperature conditions in the short, mid, and long term.Results showed that, in the studied area, the bathymetric distribution of W. setacea is restricted to a depth belt between 25 and 40 m deep, reaching maximum biomass values (126 g dw m−2) at 30 m depth. In concordance, although in the short term W. setacea survived and grew in a large range of environmental conditions, its life requirements for the mid and long term were dim light levels and low temperatures. Biomass of Womersleyella setacea did not show any clear seasonal pattern, though minimum values were reported in spring. Reproductive structures were always absent. Bearing in mind that no herbivores feed on Womersleyella setacea and that its thermal preferences are more characteristic of temperate than of tropical seaweeds, low light (50 µmol photon m−2 s−1) and low temperature (12°C) levels are critical for W. setacea survival and growth, thus probably determining its spread and bathymetric distribution across the Mediterranean Sea