Showing papers by "University of Leicester published in 1996"

Getting the glycosylation right: implications for the biotechnology industry.

Abstract: Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

Emotions are social.

Abstract: In this paper, I question the assumption that emotions are first and foremost individual reactions, and suggest instead that they are often best viewed as social phenomena. I show that many of the causes of emotions are interpersonally, institutionally or culturally defined; that emotions usually have consequences for other people; and that they serve interpersonal as well as cultural functions in everyday life. Furthermore, many cases of emotion are essentially communicative rather than internal and reactive phenomena. Previous research has often underestimated the importance of social factors in the causation and constitution of emotion. In conclusion, I recommend that existing cognitive and physiological approaches to emotional phenomena be supplemented or supplanted by social psychological analysis.

Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.

Abstract: Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies

Abstract: Unlike structures determined by X-ray crystallography, which are deposited in the Brookhaven Protein Data Bank (Abola et al., 1987) as a single structure, each NMR-derived structure is often deposited as an ensemble containing many structures, each consistent with the restraint set used. However, there is often a need to select a single 'representative' structure, or a 'representative' subset of structures, from such an ensemble. This is useful, for example, in the case of homology modelling or when compiling a relational database of protein structures. It has been shown that cluster analysis, based on overall fold, followed by selection of the structure closest to the centroid of the largest cluster, is likely to identify a structure more representative of the ensemble than the commonly used minimized average structure (Sutcliffe, 1993). Two approaches to the problem of clustering ensembles of NMR-derived structures have been described. One of these (Adzhubei et al., 1995) performs the pairwise superposition of all structures using C a atoms to generate a set of r.m.s. distances. After cluster analysis based on these distances, a user-defined cut-off is required to determine the final membership of clusters and therefore the representative structures. The other approach (Diamond, 1995) uses collective superpositions and rigid-body transformations. Again, the position at which to draw a cut-off based on the particular clustering pattern was not addressed. Whenever fixed values are used for the cut-off in clustering, there is a danger of missing 'true' clusters under the threshold imposed by the rigid cut-off value. Considering the highly diverse nature of NMR-derived ensembles of proteins, it would seem most appropriate to avoid the use of predefined values for determining clusters. In fact, of the 302 ensembles we have studied, the average pairwise r.m.s. distance across an ensemble varied from 0.29 to 11.3 A (mean value 3.0, SD 1.9 A). Here we present an automated method for cut-off determination that avoids the dangers of using fixed values for this purpose. We have developed a computer program that automatically, systematically and rapidly (i) clusters an ensemble of structures into a set of conformationally related subfamilies, and (ii) selects a representative structure from each cluster. The program uses the method of average linkage to define how clusters are built up, followed by the application of a penalty function that seeks to minimize simultaneously the number of clusters

Actuator fault diagnosis: an adaptive observer-based technique

Abstract: This paper presents a novel approach for the fault diagnosis of actuators in known deterministic dynamic systems by using an adaptive observer technique. Systems without model uncertainty are initially considered, followed by a discussion of a general situation where the system is subjected to either model uncertainty or external disturbance. Under the assumption that the system state observer can be designed such that the observation error is strictly positive real (SPR), an adaptive diagnostic algorithm is developed to diagnose the fault, and a modified version is proposed for the general system to improve robustness. The method is demonstrated through its application to a simulated second-order system.

A meta-analysis of the association of the deletion allele of the angiotensin-converting enzyme gene with myocardial infarction.

Abstract: Background The ACE gene is characterized by a polymorphism based on the presence (insertion [I]) or absence (deletion [D]) within intron 16 of a 287-basepair alu repeat sequence, resulting in three genotypes. Subsequent studies have produced conflicting findings. To further evaluate the association of the ACE I/D genotype with MI risk, we carried out a meta-analysis of all the published studies. Methods and Results In total, 15 studies containing 3394 MI cases and 5479 control subjects were analyzed. The overall distribution of genotypes in the control subjects was 22.7% II, 49.0% ID, and 28.3% DD. The mean odds ratio for MI for DD versus ID/II genotypes across all studies was 1.26 (95% CI, 1.15, 1.39; P<.0001). Pairwise odds ratios were 1.36 (95% CI, 1.19, 1.55) for DD and II, 1.24 (95% CI, 1.11, 1.38) for DD and ID, and 1.09 (95% CI, 0.96, 1.23) for ID and II. The relative risk appeared to be increased in Japanese populations (2.55; 95% CI, 1.75, 3.70). Conclusions Within the limitations of the availabl...

Presynaptic calcium current modulation by a metabotropic glutamate receptor.

Abstract: Metabotropic glutamate receptors (mGluRs) regulate transmitter release at mammalian central synapses. However, because of the difficulty of recording from mammalian presynaptic terminals, the mechanism underlying mGluR-mediated presynaptic inhibition is not known. Here, simultaneous recordings from a giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body were obtained in rat brainstem slices. Agonists of mGluRs suppressed a high voltage-activated P/Q-type calcium conductance in the presynaptic terminal, thereby inhibiting transmitter release at this glutamatergic synapse. Because several forms of presynaptic modulation and plasticity are mediated by mGluRs, this identification of a target ion channel is a first step toward elucidation of their molecular mechanism.

Discovery of X-ray and Extreme Ultraviolet Emission from Comet C/Hyakutake 1996 B2

Abstract: During its close approach to Earth, comet C/Hyakutake 1996 B2 was observed at extreme ultraviolet and x-ray wavelengths with the Rœntgen X-ray Satellite and Rossi X-ray Timing Explorer. The emission morphology was symmetric with respect to a vector from the comet's nucleus toward the sun, but not symmetric around the direction of motion of the comet with respect to interplanetary dust. A slowly varying emission and a large impulsive event that varied on time scales of 1 to 2 hours were observed. An interaction between the comet and the solar wind/solar magnetic field seems to be the most likely mechanism for the observed emission.

Human minisatellite mutation rate after the Chernobyl accident

Abstract: Germline mutation at human minisatellite loci has been studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation was found to be twice as high in the exposed families as in the control group. Mutation rate in the Mogilev families was correlated with the level of caesium-137 surface contamination, consistent with radiation induction of germline mutation.

Crystal structure of a PDZ domain

Abstract: PDZ domains (also known as DHR domains or GLGF repeats) are ~90-residue repeats found in a number of proteins implicated in ion-channel and receptor clustering, and the linking of receptors to effector enzymes1. PDZ domains are protein-recognition modules; some recognize proteins containing the consensus carboxy-terminal tripeptide motif S/TXV with high specificity2–4. Other PDZ domains form homotypic dimers: the PDZ domain of the neuronal enzyme nitric oxide synthase binds to the PDZ domain of PSD-95, an interaction that has been implicated in its synaptic association5. Here we report the crystal structure of the third PDZ domain of the human homologue of the Drosophila discs-large tumour-suppressor gene product, DlgA. It consists of a five-stranded antiparallel β-barrel flanked by three α-helices. A groove runs over the surface of the domain, ending in a conserved hydrophobic pocket and a buried arginine; we suggest that this is the binding site for the C-terminal peptide.

Migration theories and evidence: an assessment

Abstract: . This paper presents a critical survey of theories of migration, their welfare and policy implications and their empirical relevance. We also develop some extensions to the theory beginning with the Harris and Todaro (HT) model. In particular, the HT model is extended to examine risk averse behaviour within families where the migration of members of families serves to diversify risk. The welfare implications of the individual migration decision and government intervention in the form of employment subsidies are examined. Recent evidence on international migration is presented. It is shown that migration does not flow automatically in response to wage differentials. Characteristics of migrants and the process of self-selection are found to be important determinants of the rate of migration.

Calcium signalling in bacteria

Abstract: Copyright © 1996, American Society for Microbiology. Also available from http://jb.asm.org/cgi/reprint/178/13/3677

A prospective study of self-esteem in the prediction of eating problems in adolescent schoolgirls: Questionnaire findings

Abstract: A number of authors have emphasized the importance of self-esteem in the aetiology of the eating disorders anorexia nervosa and bulimia nervosa. Evidence for such theorizing, however, mainly derives from clinical observations on people being treated for eating disorders. This study is the first prospective study to investigate the role of self-esteem in aetiology prior to the onset of an eating disorder. Self-esteem was measured in 594 schoolgirls aged 11-12 using the Rosenberg Self-Esteem Scale (Rosenberg, 1965). Almost 400 of these girls were successfully followed up at age 15-16 and they completed a questionnaire examining eating and other psychological problems. Results showed that girls with low self-esteem at age 11-12 were at significantly greater risk of developing the more severe signs of eating disorders, as well as other psychological problems, by the age of 15-16. It is argued that more research is needed to replicate and extend these findings. The results also give weight to the case for examining the potential role of self-esteem enhancement in the prevention of eating disorders.

Regulation of translation elongation factor‐2 by insulin via a rapamycin‐sensitive signalling pathway.

Abstract: It is well established that insulin and serum stimulate gene expression at the level of mRNA translation in animal cells, and previous studies have mainly focused on the initiation process. Here we show that, in Chinese hamster ovary cells expressing the human insulin receptor, insulin causes decreased phosphorylation of elongation factor eEF-2 and that this is associated with stimulation of the rate of peptide-chain elongation. eEF-2 is phosphorylated by a very specific Ca 2+/calmodulin-dependent protein kinase (eEF-2 kinase) causing its complete inactivation. The decrease in eEF-2 phosphorylation induced by insulin reflects a fall in eEF-2 kinase activity. Rapamycin, a macrolide immunosuppressant which blocks the signalling pathway leading to the stimulation of the 70/85 kDa ribosomal protein S6 kinases, substantially blocks the activation of elongation, the fall in eEF-2 phosphorylation and the decrease in eEF-2 kinase activity, suggesting that p7O S6 kinase (p70s6k) and eEF-2 kinase may tie on a common signalling pathway. Wortmannin, an inhibitor of phosphatidylinositide-3-OH kinase, had similar effects. eEF-2 kinase was phosphorylated in vitro by purified p70s6k but this had no significant effect on the in vitro activity of eEF-2 kinase.

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site

Abstract: We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102–497, 951–1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Cation-pi bonding and amino-aromatic interactions in the biomolecular recognition of substituted ammonium ligands.

Abstract: Cation-π bonds and amino-aromatic interactions are known to be important contributors to protein architecture and stability, and their role in ligand-protein interactions has also been reported. Many biologically active amines contain substituted ammonium moieties, and cation-π bonding and amino-aromatic interactions often enable these molecules to associate with proteins. The role of organic cation-π bonding and amino-aromatic interactions in the recognition of small-molecule amines and peptides by proteins is an important topic for those involved in structure-based drug design, and although the number of structures determined for proteins displaying these interactions is small, general features are beginning to emerge. This review explores the role of cation-π bonding and amino-aromatic interactions in the biological molecular recognition of amine ligands. Perspectives on the design of ammonium-ligand-binding sites are also discussed.

The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin

Abstract: In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actinfilament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-l (residues 839-850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack

Abstract: Salicylic acid (SA) is absolutely required for establishment of acquired resistance in non-infected tissues following localized challenge of other leaves with a necrotizing pathogen. Although not directly responsive to SA, or induced systemically following pathogen challenge, the expression of defence gene promoter fusions AoPR1—GUS and PAL-3—GUS after wounding or pathogen challenge could be enhanced by pre-treating tobacco plants hydroponically with SA, a phenomenon designated ‘potentiation’. Potentiation of AoPR1—GUS wound-responsiveness was also demonstrated locally, but not systemically, in tobacco tissue exhibiting acquired resistance following infection with either viral or bacterial pathogens. Potentiation of wound-responsive expression by prior wounding could not be demonstrated. In contrast, potentiation of pathogen-responsive AoPR1—GUS expression was exhibited both locally and systemically in non-infected tissue. The spatial and temporal exhibition of defence gene potentiation correlated directly with the acquisition of resistance in non-infected tissue. Pathogen-responsive potentiation was obtained at about 10-fold lower levels of salicylic acid than wounding-responsive potentiation in AoPR1—GUS tobacco plants prefed with salicylate. These results may explain the failure to observe systemic potentiation of the wound-responsive defence gene expression. The data suggest a dual role for SA in terms of gene induction in acquired immunity: a direct one by induction of genes such as pathogenesis-related proteins, and an indirect one by potentiation of expression of other local defence genes (such as PAL and AoPR1) which do not respond directly to SA but become induced on pathogen attack or wounding.

Breast carcinomas occurring in young women (< 35 years) are different

Abstract: One hundred and sixty-three breast carcinomas occurring in women aged between 26 and 44 years were examined for pathological features, oestrogen and progesterone receptor status, proliferation as determined by Ki-67 labelling and the presence of c-erbB-2 and p53 protein, and were compared with a control group of carcinomas from women in the 50-67 years age group. Carcinomas occurring in women aged under 35 years had a significantly high incidence of being poorly differentiated and of having high proliferation rates. This group also had a significantly high incidence of p53 protein staining. Carcinomas in the under 30 years age group had a lower incidence of oestrogen and progesterone receptor positivity. No differences were found in c-erbB-2-positive staining between the groups. Infiltrating lobular carcinomas were only identified in women aged 40 years and over. There was a higher incidence of a family history in the 35-44 years age group (18%) than in the under 35 years age group (11%). Breast carcinomas occurring in women aged under 35 years are more aggressive. An important finding is the high incidence of p53 positivity, which may indicate genetic instability.

Black Hole Binaries and X-Ray Transients

Abstract: We consider transient behavior in low-mass X-ray binaries (LMXBs) We show that if this results from a disk instability, the secondary star must be significantly evolved when mass transfer starts, particularly if the primary is a neutron star For P 2 days, most neutron star systems will be persistent X-ray sources, whereas the slower orbital evolution of black hole systems means that most of them are likely to be transient Both types of transient system must have extreme mass ratios (<01) For orbital periods P 2 days, most LMXBs will be transient regardless of whether the primary is a neutron star or a black hole

Guanine radical cations are precursors of 7,8-dihydro-8-oxo-2'-deoxyguanosine but are not precursors of immediate strand breaks in DNA

Abstract: Biphotonic photoionization of frozen aqueous solutions of DNA at 248 nm has been shown by EPR spectroscopy to lead selectively to the guanine cation. In H218O under these conditions high levels of ...

Genetic engineering of harvest index in tobacco through overexpression of a phytochrome gene

Abstract: The phytochrome photoreceptor family regulates plant architecture in response to environmental light signals. Phytochromes mediate the shade avoidance syndrome, in which plants react to far-red radiation reflected from neighbors by elongation growth, occurring at the expense of leaf and storage organ production. We show that transgenic overproduction of phytochrome A in tobacco suppresses shade avoidance, causing proximity-conditional dwarfing. At high densities in the field, assimilates show an enhanced allocation to leaves, with a concomitant increase in harvest index. Transfer of this approach to other crop plants could provide significant improvements in productivity.

Minisatellite diversity supports a recent African origin for modern humans

Abstract: In a study of human diversity at a highly variable locus, we have mapped the internal structures of tandem–repetitive alleles from different populations at the minisatellite MS205 (D76S309).The results give an unusually detailed view of the different allelic structures represented on modern human chromosomes, and of the ancestral relationships between them. There was a clear difference in allelic diversity between African and non–African populations. A restricted set of allele families was found in non–African populations, and formed a subset of the much greater diversity seen on African chromosomes. The data strongly support a recent African origin for modern human diversity at this locus.

Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol

Abstract: Mammalian proteasomes are composed of 14‐17 dierent types of subunits, some of which, including major-histocompatibilitycomplex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol) Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt In the microsomes, proteasomes were on the outside of the membranes Further subfractionation of the microsomes showed that the proteasomes

Effects of the BKCa channel activator, NS1619, on rat cerebral artery smooth muscle.

Abstract: 1. We have investigated the actions of NS1619, a putative activator of large conductance calcium-activated potassium channels (BKCa) by use of the patch-clamp technique on smooth muscle cells enzymatically isolated from the rat basilar artery. 2. Using whole cell current-clamp to measure membrane potential, addition of 30 microM NS1619 produced cellular hyperpolarization, moving the membrane potential towards the calculated equilibrium potential for potassium. This hyperpolarization was rapidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3. In whole cell recordings made from cells voltage-clamped at 0 mV using the perforated-patch technique, addition of NS1619 (10-30 microM) activated an outward current, which reversed following washout of NS1619. 4. This outward current was unaffected by application of either glibenclamide (5 microM), an inhibitor of ATP-sensitive potassium channels, or apamin (100 nM), an inhibitor of small-conductance calcium-activated potassium channels. However, this current was almost completely abolished by iberiotoxin (IbTX; 50-100nM). 5. Depolarizing voltage steps activated small outward currents from cells held at -15 mV. Application of NS1619 (10-30 microM) increased the size of these currents, producing a shift to the left of the current-voltage (I-V) relationship. These currents were largely inhibited by IbTX (100 nM). 6. Measurements of the unitary amplitude of the single channels activated by NS1619 which could be resolved in whole cell recordings yielded a value of 5.6 +/- 0.14 pA at 0 mV. 7. NS1619 (10-30 microM) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 microM) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation produced by NS1619 was calcium-dependent and inhibited by external IbTX (100 nM). The unitary current amplitude was unaffected by NS1619. 8. By use of conventional whole cell recording methods and conditions that suppressed BKCa openings, outward potassium currents were activated by depolarizing potentials positive to -35 mV from a holding potential of -65 mV. NS1619 (10-30 microM) inhibited this current in a concentration-dependent manner. This inhibition was reversed following washout of NS1619, recovering to 60-90% of control values within 2 min. 9. Ba2+ currents, measured by conventional whole cell recording, were activated by depolarizing voltage steps from negative holding potentials. NS1619 (1-30 microM) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 microM with a Hill coefficient approaching unity. This inhibition was reversible, with the currents recovering to 65-100% of control values after washout of NS1619 for 2 min. 10. NS1619 (0.3-100 microM) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50 = 12.5 +/- 2.0 microM; n = 4). This relaxation curve was shifted to the right, but not abolished, when the tissue was treated with a blocker of BKCa channels (IbTX; 100nM). Additionally, NS1619 produced concentration-dependent relaxation of basilar artery contracted with a depolarizing, isotonic salt solution containing 80 mM K+. 11. Thus NS1619 produces hyperpolarization of basilar artery myocytes through direct activation of BKCa and also directly inhibits Ca2+ currents and voltage-activated K+ channels. We discuss the implications of these results for its vasorelaxant actions.