Showing papers by "University of Dundee published in 1998"

Peer Assessment Between Students in Colleges and Universities

Abstract: A definition and typology of peer assessment between students in higher education is proposed, and the theoretical underpinnings of the method are discussed. A review of the developing literature follows, including both process and outcome studies. This indicates that peer assessment is of adequate reliability and validity in a wide variety of applications. Peer assessment of writing and peer assessment using marks, grades, and tests have shown positive formative effects on student achievement and attitudes. These effects are as good as or better than the effects of teacher assessment. Evidence for such effects from other types of peer assessment (of presentation skills, group work or projects, and professional skills) is, as yet, more limited. Computer-assisted peer assessment is an emerging growth area. Important factors in successful implementation are summarized, and recommendations for future research and practice are made.

The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?

Abstract: Mammalian AMP-activated protein kinase and yeast SNF1 protein kinase are the central components of kinase cascades that are highly conserved between animals, fungi, and plants. The AMP-activated protein kinase cascade acts as a metabolic sensor or "fuel gauge" that monitors cellular AMP and ATP levels because it is activated by increases in the AMP:ATP ratio. Once activated, the enzyme switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways, such as fatty acid oxidation. The SNF1 complex in yeast is activated in response to the stress of glucose deprivation. In this case the intracellular signal or signals have not been identified; however, SNF1 activation is associated with depletion of ATP and elevation of AMP. The SNF1 complex acts primarily by inducing expression of genes required for catabolic pathways that generate glucose, probably by triggering phosphorylation of transcription factors. SNF1-related protein kinases in higher plants are likely to be involved in the response of plant cells to environmental and/or nutritional stress.

International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors

Abstract: Actions of acetylcholine in the periphery are the result of activation of either the ionotropic nicotinic receptor or the metabotropic muscarinic receptor. In the mammalian central nervous system (CNS)c, both nicotinic and muscarinic receptor subtypes are present on neurons, although there is as yet

Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB

Abstract: We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.

Structure and Function in the Nucleus

Abstract: Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions

Abstract: The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

Soil microbial community structure: Effects of substrate loading rates

Abstract: A fuller understanding of the interactions which affect rhizosphere microbial community structure requires experimental manipulation of the individual components of that interaction (e.g. amount and composition of exudate, soil moisture and soil nutrient status). We describe an experiment where a synthetic root exudate was applied continuously to a soil held at constant water potential. The solution contained compounds characteristic of root exudates (fructose, glucose, sucrose, succinic acid, malic acid, arginine, serine and cysteine), which were added at a range of concentrations. After 14 d of such substrate addition, a central portion of soil, known to be influenced by the added substrate, was removed for analysis. Microbial community structure of this central core was determined by the broad-scale measurements; community DNA hybridisation and %G+C profiling, and phospholipid–fatty acid analysis (PLFA). The trend was that microbial community structure changed consistently as substrate loading increased, and that fungi dominated over bacteria at high substrate loading rates. The DNA and the PLFA analyses both indicated that there was a coherent gradient of changes with increased substrate loading. This may have arisen as a consequence of the competitive ability of soil microorganisms being dependent on the quantity of available substrate.

The twelfth Tansley Lecture. Small is beautiful: the picophytoplankton

Abstract: 1. Picophytoplankton are planktonic photosynthetic O2-evolvers that can pass through 2 μm-diameter pores; they include prokaryotic (eubacterial) and eukaryotic members and occur in freshwater and marine habitats. There are no photosynthetic reproductive and dispersal stages of benthic macrophytes of picoplanktonic size. The picophytoplankton condition appears to be derived and polyphyletic in both prokaryotes and eukaryotes. 2. Picophytoplankton are among the smallest free-living cells, despite having to contain the photosynthetic apparatus, which occupies about half of the cell volume, as well as core cellular machinery. The size of the smallest prokaryotic (0·6 μm diameter) and eukaryotic (0·95 μm diameter) picophytoplankton are close to the minimum possible size estimated from the occurrence of non-scalable essential components such as the genome and plasmalemma and other membranes. 3. Picophytoplankton cells have advantages relative to larger phytoplankton cells in terms of resource acquisition and the subsequent use of the resources in catalysing cell growth and reproduction. The smaller package effect in light harvesting means smaller resource (energy, C, N, Fe, Mn, Cu) costs of photon harvesting and transformation into chemical energy in small than in large cells. The smaller diffusion boundary layer around small cells, coupled with smaller nutrient fluxes per unit plasmalemma area needed to attain a given fraction of the maximum specific growth rate in smaller cells, increases the availability of low concentrations of nutrients to small relative to larger cells. If the supply of CO2 to the core photosynthetic carboxylase ribulose bisphosphate carboxylase-oxygenase is purely by diffusion then smaller cells could satisfy their catalytic requirements with less of this enzyme whose synthesis has high energy, C and N costs. Overall, resources can be acquired, and used in growth, more effectively in smaller than in larger cells. 4. Some factors work against the conclusion in (3.) but, in most habitats, do not negate these conclusions. Examples related to non-scalable essential cell components are the use of energy, C, N and P in the genome and of energy, C, N, P and Fe in the plasma membrane. Transport-related factors include the increased potential cell volume-specific leakage of accumulated resources, and the greater cell volume-specific energy costs of motility at a given speed, in smaller than in larger cells. The smaller package effect in smaller cells involves a greater potential for photodamage by both photosynthetically active radiation and by UV-B. 5. Picophytoplankton occurrence is also a function of factors which lead to cell loss. Factors such as sinking out of the euphotic zone and parasitism by eukaryotes such as chytrids are less significant for picophytoplankton than for larger cells, whereas viral parasitism and grazing by appropriately sized grazers are likely to be at least as great for picophytoplankton as for larger cells. 6. The totality of the effects mentioned in (3.) above suggests that picophytoplankton should generally have higher specific growth rates (probably) in resource-saturated (photons, C, N, P, Fe, etc.) and (certainly) in resource-limited environments than do larger cells. The distribution of picophytoplankton is certainly consistent with their ability to capitalize on resource-limited environments: they contribute a larger fraction of biomass and productivity relative to larger cells in low-nutrient than in high-nutrient environments, and in low-light (e.g. the deep chlorophyll maximum) than in high-light environments. 7. Sexual reproduction is apparently rare in picophytoplankton, with implications for species definition. There are probably relatively few species (hundreds or thousands) with very wide biogeographical ranges in marine or in freshwater habitats. The small size and (probably) low biodiversity means large numbers of individuals worldwide, e.g. c.1026 individuals of the commonest species of the marine cyanobacterium Synechococcus. Picophytoplankton contribute at least one-tenth (i.e. in excess of 3 Pg C per year) to global aquatic net primary productivity.

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.

Abstract: Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Polo-like kinases: a team that plays throughout mitosis

Abstract: When the first mutant allele of the Drosophila genepolo was first characterized over 10 years ago, attention focused on the defects that centrosome behavior exhibited at various stages of development (Sunkel and Glover 1988). The subsequent realization that the serine-threonine kinase it encodes is highly conserved from yeasts to humans has provoked a flurry of investigation into the function of the enzyme. A role for the polo-like kinases (plks) in regulating centrosome behavior has been borne out in several organisms, and the enzymes have attracted further attention recently with the realization that they regulate multiple stages of mitotic progression. In this article we review the current status of our understanding of the functions of plks from the time of commitment to M phase in the activation of Cdc25, through the activation of the anaphase promoting complex (APC), to the regulation of late mitotic events essential for cytokinesis. We discuss how to reconcile the sometimes apparently disparate observations made upon plk function in different organisms.

AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform.

Abstract: Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric alphabetagamma complexes, where the catalytic subunit has two isoforms (alpha1 and alpha2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the alpha2 rather than the alpha1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein-alpha-subunit fusions. By all three methods a significant proportion of alpha2, but not alpha1, is localized in the nucleus. Like SNF1, AMPK-alpha2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of alpha1 and alpha2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Abstract: Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophy...

AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release.

Abstract: The role of the AMP-activated protein kinase (AMPK) cascade in the glucose-sensitive pancreatic beta cell lines HIT-T15 and INS-1 was addressed. In both cell types, removal of glucose leads to a >5-fold activation of AMPK activity. Activation of AMPK was due to phosphorylation, since the effect was reversed by protein phosphatase treatment of the extracts, and was restored by re-addition of MgATP and the purified upstream kinase. When the effects of different concentrations of medium glucose were examined, insulin secretion and AMPK activity were inversely related, and varied over the same concentration range. The activation in response to glucose removal appeared to be due to changes in the concentration of the known regulators of the cascade, i.e. AMP and ATP, since AMPK activation was associated with a large increase in the cellular AMP/ATP ratio, and the two parameters varied over the same range of glucose concentrations. In late-passage HIT-T15 cells that had lost the glucose-dependent insulin secretion response, both AMPK activity and the AMP/ATP ratio also became insensitive to the extracellular glucose concentration. Treatment of INS-1 cells, but not HIT-T15 cells, with AICA riboside (5-aminoimidazole-4-carboxamide riboside) results in accumulation of the ribotide, ZMP (AICA riboside monophosphate), and activation of AMPK. AICA riboside treatment of INS-1 cells, and of isolated rat islets, had both inhibitory and stimulatory effects on insulin secretion. These results show that in beta cell lines the AMP-activated protein kinase, like its yeast homologue the SNF1 complex, can respond to the level of glucose in the medium, and may be involved in regulating insulin release.

pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis

Abstract: Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. We show that the septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV‐KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. We show that Polo kinase associates with PAV‐KLP with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pav mutants. We suggest that PAV‐KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins.

PCNA binding through a conserved motif.

Abstract: Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of several proteins. The cell cycle regulatory protein, p21, and the replication endonuclease, Fen1, have already been described as competing for PCNA binding. Two recent reports have identified DNA (cytosine-5)methyltransferase (MCMT) and the DNA repair endonuclease XPG as binding to PCNA.1,2 The remarkable thing about these interactions is that they all seem to occur through a conserved motif that is likely to contact the same site on PCNA. This has fascinating implications for a regulatory network linking these diverse protein functions. BioEssays20:195–199, 1998. © 1998 John Wiley & Sons, Inc.

An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation

Abstract: Foreign protein antigens must be broken down within endosomes or lysosomes to generate suitable peptides that will form complexes with class II major histocompatibility complex molecules for presentation to T cells. However, it is not known which proteases are required for antigen processing. To investigate this, we exposed a domain of the microbial tetanus toxin antigen (TTCF) to disrupted lysosomes that had been purified from a human B-cell line. Here we show that the dominant processing activity is not one of the known lysosomal cathepsins, which are generally believed to be the principal enzymes involved in antigen processing, but is instead an asparagine-specific cysteine endopeptidase. This enzyme seems similar or identical to a mammalian homologue of the legumain/haemoglobinase asparaginyl endopeptidases found originally in plants and parasites. We designed competitive peptide inhibitors of B-cell asparaginyl endopeptidase (AEP) that specifically block its proteolytic activity and inhibit processing of TTCF in vitro. In vivo, these inhibitors slow TTCF presentation to T cells, whereas preprocessing of TTCF with AEP accelerates its presentation, indicating that this enzyme performs a key step in TTCF processing. We also show that N-glycosylation of asparagine residues blocks AEP action in vitro. This indicates that N-glycosylation could eliminate sites of processing by AEP in mammalian proteins, allowing preferential processing of microbial antigens.

SNF1-related protein kinases: global regulators of carbon metabolism in plants?

Abstract: The SNF1 protein kinase family currently comprises SNF1 itself in the yeast Saccharomyces cerevisiae, the AMP-activated protein kinases (AMPK) in mammals, and the SNF1-related protein kinases (SnRKs) in higher plants Members of the family have been discovered and rediscovered several times in recent years by different biochemical assays and/or genetic screens, and only when DNA and amino acid sequences became available was it realised that all of the different functions described were due to the members of the same protein kinase family The physiological roles of the SNF1 family are currently better defined in yeast and animals, so it is necessary to begin our review with a description of those systems However, some of the higher-plant SnRKs appear to be highly conserved with their yeast and animal counterparts, and we suspect they will turn out to play very similar roles From the yeast and animal studies described in more detail below, the concept is emerging that the SNF1 family protect cells against nutritional or environmental stresses, particularly those which compromise cellular energy status, by regulating both metabolism and gene expression

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.

Abstract: Entry into anaphase and exit from mitosis depend on a ubiquitin–protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin–protein ligase complex SCFCdc4 required for the initiation of DNA replication.

Amino acid availability regulates p70 S6 kinase and multiple translation factors.

Abstract: Incubation of Chinese hamster ovary cells without amino acids for up to 60 min caused a rapid marked decrease in p70 S6 kinase activity and increased binding of initiation factor eIF4E to its inhibitory regulator protein 4E-BP1. This was associated with dephosphorylation of 4E-BP1 and eIF4E and dissociation of eIF4E from eIF4G. All these effects were rapidly reversed by resupplying a mixture of amino acids and this was blocked by rapamycin and by inhibitors of phosphatidylinositol 3-kinase, implying a role for phosphatidylinositol 3-kinase in the signalling pathway linking amino acids with the control of p70 S6 kinase activity and the phosphorylation of these translation factors. Amino acid withdrawal also led to changes in the phosphorylation of other translation factors; phosphorylation of eIF4E decreased whereas elongation factor eEF2 became more heavily phosphorylated, each of these changes being associated with decreased activity of the factor in question. Earlier studies have suggested that protein kinase B (PKB) may act upstream of p70 S6 kinase. However, amino acids did not affect the activity of PKB, indicating that amino acids activate p70 S6 kinase through a pathway independent of this enzyme. Studies with individual amino acids suggested that the effects on p70 S6 kinase activity and translation-factor phosphorylation were independent of cell swelling. The data show that amino acid supply regulates multiple translation factors in mammalian cells.