Showing papers by "University of Dundee published in 2004"

PRODRG: a tool for high-throughput crystallography of protein–ligand complexes

Abstract: The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein–ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-­PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

Abstract: We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

Constructing questionnaires based on the theory of planned behaviour: A manual for health services researchers

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LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

Abstract: We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.

The AMP-activated protein kinase pathway – new players upstream and downstream

Abstract: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy status Whenever the cellular ATP:ADP ratio falls, owing to a stress that inhibits ATP production or increases ATP consumption, this is amplified by adenylate kinase into a much larger increase in the AMP:ATP ratio AMP activates the system by binding to two tandem domains on the gamma subunits of AMPK, and this is antagonized by high concentrations of ATP AMP binding causes activation by a sensitive mechanism involving phosphorylation of AMPK by the tumour suppressor LKB1 Once activated, AMPK switches on catabolic pathways that generate ATP while switching off ATP-consuming processes As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of hormones and cytokines such as leptin, adiponectin and ghrelin A particularly interesting downstream target recently identified is TSC2 (tuberin) The LKB1-->AMPK-->TSC2 pathway negatively regulates the target of rapamycin (TOR), and this appears to be responsible for limiting protein synthesis and cell growth, and protecting against apoptosis, during cellular stresses such as glucose starvation

The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins.

Abstract: Insulin-like growth factors elicit many responses through activation of phosphoinositide 3-OH kinase (PI3K). The tuberous sclerosis complex (TSC1-2) suppresses cell growth by negatively regulating a protein kinase, p70S6K (S6K1), which generally requires PI3K signals for its activation. Here, we show that TSC1-2 is required for insulin signaling to PI3K. TSC1-2 maintains insulin signaling to PI3K by restraining the activity of S6K, which when activated inactivates insulin receptor substrate (IRS) function, via repression of IRS-1 gene expression and via direct phosphorylation of IRS-1. Our results argue that the low malignant potential of tumors arising from TSC1-2 dysfunction may be explained by the failure of TSC mutant cells to activate PI3K and its downstream effectors.

Irrigation scheduling: advantages and pitfalls of plant-based methods.

Abstract: This paper reviews the various methods available for irrigation scheduling, contrasting traditional water-balance and soil moisture-based approaches with those based on sensing of the plant response to water deficits. The main plant-based methods for irrigation scheduling, including those based on direct or indirect measurement of plant water status and those based on plant physiological responses to drought, are outlined and evaluated. Specific plant-based methods include the use of dendrometry, fruit gauges, and other tissue water content sensors, while measurements of growth, sap flow, and stomatal conductance are also outlined. Recent advances, especially in the use of infrared thermometry and thermography for the study of stomatal conductance changes, are highlighted. The relative suitabilities of different approaches for specific crop and climatic situations are discussed, with the aim of indicating the strengths and weaknesses of different approaches, and highlighting their suitability over different spatial and temporal scales. The potential of soil- and plant-based systems for automated irrigation control using various scheduling techniques is also discussed.

Interactions and self-organization in the soil-microbe complex.

Abstract: Soil is the most complicated biomaterial on the planet. As with any material, the physical habitat is of prime importance in determining and regulating biological activity. However, until recently the opaque nature of soil has meant that any interrogation of its interior architecture has been relatively rudimentary, restricted to simple qualitative expressions of the physical heterogeneity that fail to relate to any specific function. However, new techniques and insights into the biophysical and biochemical processes of this inner space are leading to the developments of theoretical frameworks and experimental approaches that will allow us to sustainably manage Earth's most important resource. We introduce the concept that the soil-microbe system is self-organized and suggest new priorities for research based on an integrative approach that combines biochemistry and biophysics.

GSK3 inhibitors: development and therapeutic potential

Abstract: Glycogen synthase kinase-3 (GSK3) was initially identified more than two decades ago as an enzyme involved in the control of glycogen metabolism. In recent years it has been shown to have key roles in regulating a diverse range of cellular functions, which have prompted efforts to develop GSK3 inhibitors as therapeutics. Here, we describe the biology of GSK3 relevant to its potential as a target for diabetes and neurodegenerative diseases, and discuss progress in the development of GSK3 inhibitors.

Focus group interviews as a data collecting strategy

Abstract: Background. Focus group interviews are a method for collecting qualitative data and have enjoyed a surge in popularity in health care research over the last 20 years. However, the literature on this method is ambiguous in relation to the size, constitution, purpose and execution of focus groups. Aim. The aim of this article is to explore some of the methodological issues arising from using focus group interviews in order to stimulate debate about their efficacy. Discussion. Methodological issues are discussed in the context of a study examining attitudes towards and beliefs about older adults in hospital settings among first-level registered nurses, nursing lecturers and student nurses. Focus group interviews were used to identify everyday language and constructs used by nurses, with the intention of incorporating the findings into an instrument to measure attitudes and beliefs quantitatively. Conclusions. Experiences of conducting focus group interviews demonstrated that smaller groups were more manageable and that groups made up of strangers required more moderator intervention. However, as a data collecting strategy they are a rich source of information.

CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations

Abstract: CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (γ2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine β-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine β-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.

Characterization of Bacterial Communities in Feces from Healthy Elderly Volunteers and Hospitalized Elderly Patients by Using Real-Time PCR and Effects of Antibiotic Treatment on the Fecal Microbiota

Abstract: Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years; n = 35) living in the local community, elderly hospitalized patients (age, 66 to 103; n = 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100; n = 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in the Bacteroides-Prevotella group following hospitalization. Reductions in bifidobacteria, Desulfovibrio spp., Clostridium clostridiiforme, and Faecalibacterium prausnitzii were also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria, Desulfovibrio spp., C. clostridiiforme, and F. prausnitzii did not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic species Enterococcus faecalis proliferated.

European clinical guidelines for hyperkinetic disorder-first upgrade

Abstract: BACKGROUND: The validity of clinical guidelines changes over time, because new evidence-based knowledge and experience develop OBJECTIVE: Hence, the European clinical guidelines on hyperkinetic disorder from 1998 had to be evaluated and modified METHOD: Discussions at the European Network for Hyperkinetic Disorders (EUNETHYDIS) and iterative critique of each clinical analysis Guided by evidence-based information and based on evaluation (rather than metaanalysis) of the scientific evidence a group of child psychiatrists and psychologists from several European countries updated the guidelines of 1998 When reliable information is lacking the group gives a clinical consensus when it could be found among themselves RESULTS: The group presents here a set of recommendations for the conceptualization and management of hyperkinetic disorder and attention deficit/hyperactivity disorder (ADHD) CONCLUSION: A general scheme for practice in Europe could be provided, on behalf of the European Society for Child and Adolescent Psychiatry (ESCAP)

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes.

Abstract: 14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses.

Enhanced Dendritic Cell Antigen Capture via Toll-Like Receptor-Induced Actin Remodeling

Abstract: Microbial products are sensed through Toll-like receptors (TLRs) and trigger a program of dendritic cell (DC) maturation that enables DCs to activate T cells. Although an accepted hallmark of this response is eventual down-regulation of DC endocytic capacity, we show that TLR ligands first acutely stimulate antigen macropinocytosis, leading to enhanced presentation on class I and class II major histocompatibility complex molecules. Simultaneously, actin-rich podosomes disappear, which suggests a coordinated redeployment of actin to fuel endocytosis. These reciprocal changes are transient and require p38 and extracellular signal-regulated kinase activation. Thus, the DC actin cytoskeleton can be rapidly mobilized in response to innate immune stimuli to enhance antigen capture and presentation.

FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P.

Abstract: The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.

14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.

Abstract: 14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.

Intermediate filament proteins and their associated diseases.

Abstract: Intermediate filaments provide scaffolding for the cell and protect it against stress. This comprehensive review points out that there are more than 100 intermediate filament genes and that mutant intermediate filament proteins cause more than 30 diseases. Selective expression of these genes in particular tissues accounts for tissue-specific diseases caused by mutant intermediate filament genes, such as epidermolysis bullosa simplex and certain types of cardiomyopathy.

PTEN function: how normal cells control it and tumour cells lose it

Abstract: The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a PI (phosphoinositide) 3-phosphatase that can inhibit cellular proliferation, survival and growth by inactivating PI 3-kinase-dependent signalling. It also suppresses cellular motility through mechanisms that may be partially independent of phosphatase activity. PTEN is one of the most commonly lost tumour suppressors in human cancer, and its deregulation is also implicated in several other diseases. Here we discuss recent developments in our understanding of how the cellular activity of PTEN is regulated, and the closely related question of how this activity is lost in tumours. Cellular PTEN function appears to be regulated by controlling both the expression of the enzyme and also its activity through mechanisms including oxidation and phosphorylation-based control of non-substrate membrane binding. Therefore mutation of PTEN in tumours disrupts not only the catalytic function of PTEN, but also its regulatory aspects. However, although mutation of PTEN is uncommon in many human tumour types, loss of PTEN expression seems to be more frequent. It is currently unclear how these tumours lose PTEN expression in the absence of mutation, and while some data implicate other potential tumour suppressors and oncogenes in this process, this area seems likely to be a key focus of future research.

How do age and tooth loss affect oral health impacts and quality of life? A study comparing two national samples.

Abstract: – Age and loss of teeth can be expected to have a complex relationship with oral health-related quality of life. This study aimed to explain how age and tooth loss affect the impact of oral health on daily living using the short form, 14-item Oral Health Impact Profile (OHIP-14) on national population samples of dentate adults from the UK (1998 UK Adult Dental Health Survey) and Australia (1999 National Dental Telephone Interview Survey). After correcting for key covariables, increasing age was associated with better mean impact scores in both populations. Those aged 30–49 years in Australia showed the worst (highest) scores. In the UK, those aged under 30 showed the highest scores. In both countries, adults aged 70+ showed much better scores than the rest (P < 0.001). When corrected for age, the independent effect of tooth loss was that the worst scores were found where there were fewer than 17 natural teeth in the UK and fewer than 21 teeth in Australia. People with 25 or more teeth averaged much better scores than all other groups (P < 0.001), although there were differences in pattern between countries. When Australians were analysed by region of birth, the pattern of scores by tooth loss for British/Irish immigrants was strikingly similar to that for the UK sample. First-generation immigrants from elsewhere showed much worse overall scores and a profoundly different pattern to the Australian- and British-born groups. Age, number of teeth and cultural background are important variables influencing oral health-related quality of life.

Araucaria: software for argument analysis, diagramming and representation

Abstract: Argumentation theory involves the analysis of naturally occurring argument, and one key tool employed to this end both in the academic community and in teaching critical thinking skills to undergraduates is argument diagramming. By identifying the structure of an argument in terms of its constituents and the relationships between them, it becomes easier to critically evaluate each part of an argument in turn. The task of analysis and diagramming, however, is labor intensive and often idiosyncratic, which can make academic exchange difficult. The Araucaria system provides an interface which supports the diagramming process, and then saves the result using AML, an open standard, designed in XML, for describing argument structure. Araucaria aims to be of use not only in pedagogical situations, but also in support of research activity. As a result, it has been designed from the outset to handle more advanced argumentation theoretic concepts such as schemes, which capture stereotypical patterns of reasoning. The software is also designed to be compatible with a number of applications under development, including dialogic interaction and online corpus provision. Together, these features, combined with its platform independence and ease of use, have the potential to make Araucaria a valuable resource for the academic community.

Human antibody-Fc receptor interactions illuminated by crystal structures.

Abstract: Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies.

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Abstract: Inflammatory substances released by mast cells induce and maintain the allergic response1,2. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE)2,3. Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals2,3,4,5. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Control of the Size of the Human Muscle Mass

Abstract: This review is divided into two parts, the first dealing with the cell and molecular biology of muscle in terms of growth and wasting and the second being an account of current knowledge of physiological mechanisms involved in the alteration of size of the human muscle mass. Wherever possible, attempts have been made to interrelate the information in each part and to provide the most likely explanation for phenomena that are currently only partially understood. The review should be of interest to cell and molecular biologists who know little of human muscle physiology and to physicians, physiotherapists, and kinesiologists who may be familiar with the gross behavior of human muscle but wish to understand more about the underlying mechanisms of change.

Why older people do not participate in leisure time physical activity: a survey of activity levels, beliefs and deterrents

Abstract: Background Regular physical activity has been shown to have many health benefits. However, many older people are physically inactive. Objective To investigate why older people are reluctant to participate in leisure time physical activity and to identify strategies to encourage increased activity. Design Cross-sectional survey. Setting 16 general practices in Dundee, Scotland. Methods 409 randomly selected older people (65-84 years) who lived independently were interviewed at home. Forty-six percent of those invited to take part were recruited into the study. Results Levels of knowledge about the specific health benefits of physical activity were high. Almost all participants (95%) believed that physical activity was beneficial and 79% believed that they did enough to keep healthy. However, 36% did no leisure time physical activity and a further 17% did less than 2 hours per week. Regression modelling identified 11 factors that exerted significant independent effects on levels of leisure time physical activity. The most powerful deterrent was lack of interest (OR = 7.8). Other factors included lack of daily access to a car, shortness of breath, joint pain, dislike of going out alone or in the evening, perceived lack of fitness, lack of energy, doubting that exercise can lengthen life, not belonging to a group and doubting that meeting new people is beneficial. Conclusions Increasing leisure time physical activities poses major challenges. Beliefs about desirable levels of activity in older people would need to be changed. Action would be needed to relieve physical symptoms and address fears about perceived ability to undertake physical activity. Finally, easily accessible facilities would be needed to encourage participation in physical activity.