A novel RGD-independent cel adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs:implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.

Self-association of calcium-binding protein S100A4 and metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

Status and professional association councils. Available in 4 volumes.

This thesis examines the predictive value of a conceptual distinction between status-seeking associations and status-maintaining associations for enhancing understanding of ten selected professional associations and of the attitudes, values, behaviour and policies of their governing organs. Thirty four specific hypotheses have been tested by such research methods as questionnaires administered to individuals and associations, participant observation and an examination of association minutes and publications. Certain hypotheses have been found to be valid for particular matched pairs and/or groups of associations. The findings of the study suggest that the present conceptualisation of profession, the individual professional, professionalism, professionalisation, professional status and that relating to the role of the professions in society needs to be refined and modified in varying degrees in application to accounting associations, business graduate associations and management associations. The concept of the `ideal type' profession is shown to be of limited value in understanding certain aspects of the activities of business graduate and management associations. The findings of the study suggest that in future the professional associations examined may attach less importance to their qualifying role and lay more stress upon their representational role. The professional association faces a managerial challenge to adjust and adapt to a range of `external' pressures and `internal' demands from members and may increasingly need to be regarded as an organisation that possesses certain combinations or sets of characteristics rather than as a type of organisation that possesses a particular or relatively exclusive set. With a blurring of the distinction between the professional and state sector vocational education, and a growing customer/market orientation associated with the changing nature of work, membership of a professional association may, in future, come to be associated rather more with securing access to a relevant range of services and less with qualification for a particular career.

The association between the use of IP by UK SMEs in 2001 and subsequent performance in 2002 to 2004

This report analyses the 2001 cohort of UK SMEs. The specific focus is on the link between IP activity in 2001 and subsequent performance (to 2004). The 2001 cohort contains 130,082 SMEs of which 3,123 were IP active (2.4%). Specifically, 1,872 SMEs had at least one UK trade mark publication; 697 had one or more Community trade mark registrations; 646 SMEs had one or more UK patents; and 443 had one or more EPO patent publications. The outcome and financial performance of the SMEs is analysed in various ways. Initially, we look at the determinants of survival to 2004. We then look at growth of assets and turnover for the period 2001 to 2004.

The association between glycated haemoglobin levels and P100 visual evoked potentials in diabetes mellitus

Diabetes mellitus (DM) is a metabolic disorder which is characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action or both. The long-term specific effects of DM include the development of retinopathy, nephropathy and neuropathy. Cardiac disease, peripheral arterial and cerebrovascular disease are also known to be linked with DM. Type 1 diabetes mellitus (T1DM) accounts for approximately 10% of all individuals with DM, and insulin therapy is the only available treatment. Type 2 diabetes mellitus (T2DM) accounts for 90% of all individuals with DM. Diet, exercise, oral hypoglycaemic agents and occasionally exogenous insulin are used to manage T2DM. The diagnosis of DM is made where the glycated haemoglobin (HbA1c) percentage is greater than 6.5%. Pattern-reversal visual evoked potential (PVEP) testing is an objective means of evaluating impulse conduction along the central nervous pathways. Increased peak time of the visual P100 waveform is an expression of structural damage at the level of myelinated optic nerve fibres. This was an observational cross sectional study. The participants were grouped into two phases. Phase 1, the control group, consisted of 30 healthy non-diabetic participants. Phase 2 comprised of 104 diabetic participants of whom 52 had an HbA1c greater than 10% (poorly controlled DM) and 52 whose HbA1c was 10% and less (moderately controlled DM). The aim of this study was to firstly observe the possible association between glycated haemoglobin levels and P100 peak time of pattern-reversal visual evoked potentials (PVEPs) in DM. Secondly, to assess whether the central nervous system (CNS) and in particular visual function is affected by type and/or duration of DM. The cut-off values to define P100 peak time delay was calculated as the mean P100 peak time plus 2.5 X standard deviations as measured for the non-diabetic control group, and were 110.64 ms for the right eye. The proportion of delayed P100 peak time amounted to 38.5% for both diabetic groups, thus the poorly controlled group (HbA1c > 10%) did not pose an increased risk for delayed P100 peak time, relative to the moderately controlled group (HbA1c ≤ 10%). The P100 PVEP results for this study, do however, reflect significant delay (p < 0.001) of the DM group as compared to the non-diabetic group; thus, subclincal neuropathy of the CNS occurs in 38.5% of cases. The duration of DM and type of DM had no influence on the P100 peak time measurements.

The administration route is decisive for the ability of the vaccine adjuvant CAF09 to induce antigen-specific CD8+ T-cell responses:the immunological consequences of the biodistribution profile

A prerequisite for vaccine-mediated induction of CD8+ T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8+ T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been shown to result in strong CD8+ T-cell responses, whereas immunization via e.g. the intramuscular (i.m.) or subcutaneous (s.c.) routes often stimulate weak CD8+ T-cell responses. The hypothesis for this is that self-drainage of the adjuvant/antigen to the lymphoid organs, which takes place upon i.p. immunization, is required for the subsequent activation of cross-presenting lymphoid organ-resident CD8α+ DCs. In contrast, s.c. or i.m. immunization usually results in the formation of a depot at the site of injection (SOI), which hinders the self-drainage and targeting of the vaccine to cross-presenting CD8α+ DCs. We investigated this hypothesis by correlating the biodistribution pattern and the adjuvanticity of the strong CD8+ T-cell inducing liposomal cationic adjuvant formulation 09 (CAF09), which is composed of dimethyldioctadecylammonium bromide/monomycoloyl glycerol liposomes with polyinosinic:polycytidylic acid electrostatically adsorbed to the surface. Biodistribution studies with radiolabeled CAF09 and a surface-adsorbed model antigen [ovalbumin (OVA)] showed that a significantly larger fraction of the vaccine dose localized in the draining lymph nodes (dLNs) and the spleen 6 h after i.p. immunization, as compared to after i.m. immunization. Studies with fluorescently labelled OVA + CAF09 demonstrated a preferential association of OVA + CAF09 to DCs/monocytes, as compared to macrophages and B cells, following i.p. immunization. Administration of OVA + CAF09 via the i.p. route did also result in DC activation, whereas no DC activation could be measured within the same period with unadjuvanted OVA and OVA + CAF09 administered via the s.c. or i.m. routes. In the dLNs, the highest level of activated, cross-presenting CD8α+ DCs was detected at 24 h post immunization, whereas an influx of activated, migrating and cross-presenting CD103+ DCs to the dLNs could be measured after 48 h. This suggests that the CD8α+ DCs are activated by self-draining OVA + CAF09 in the lymphoid organs, whereas the CD103+ DCs are stimulated by the OVA + CAF09 at the SOI. These results support the hypothesis that the self-drainage of OVA + CAF09 to the draining LNs is required for the activation of CD8α+ DCs, while the migratory CD103+ DCs may play a role in sustaining the subsequent induction of strong CD8+ T-cell responses.

Association of SNPs in LCP1 and CTIF with hearing in 11 year old children:findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and the G-EAR consortium

BACKGROUND: The genetic basis of hearing loss in humans is relatively poorly understood. In recent years, experimental approaches including laboratory studies of early onset hearing loss in inbred mouse strains, or proteomic analyses of hair cells or hair bundles, have suggested new candidate molecules involved in hearing function. However, the relevance of these genes/gene products to hearing function in humans remains unknown. We investigated whether single nucleotide polymorphisms (SNPs) in the human orthologues of genes of interest arising from the above-mentioned studies correlate with hearing function in children. METHODS: 577 SNPs from 13 genes were each analysed by linear regression against averaged high (3, 4 and 8 kHz) or low frequency (0.5, 1 and 2 kHz) audiometry data from 4970 children in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth-cohort at age eleven years. Genes found to contain SNPs with low p-values were then investigated in 3417 adults in the G-EAR study of hearing. RESULTS: Genotypic data were available in ALSPAC for a total of 577 SNPs from 13 genes of interest. Two SNPs approached sample-wide significance (pre-specified at p = 0.00014): rs12959910 in CBP80/20-dependent translation initiation factor (CTIF) for averaged high frequency hearing (p = 0.00079, β = 0.61 dB per minor allele); and rs10492452 in L-plastin (LCP1) for averaged low frequency hearing (p = 0.00056, β = 0.45 dB). For low frequencies, rs9567638 in LCP1 also enhanced hearing in females (p = 0.0011, β = -1.76 dB; males p = 0.23, β = 0.61 dB, likelihood-ratio test p = 0.006). SNPs in LCP1 and CTIF were then examined against low and high frequency hearing data for adults in G-EAR. Although the ALSPAC results were not replicated, a SNP in LCP1, rs17601960, is in strong LD with rs9967638, and was associated with enhanced low frequency hearing in adult females in G-EAR (p = 0.00084). CONCLUSIONS: There was evidence to suggest that multiple SNPs in CTIF may contribute a small detrimental effect to hearing, and that a sex-specific locus in LCP1 is protective of hearing. No individual SNPs reached sample-wide significance in both ALSPAC and G-EAR. This is the first report of a possible association between LCP1 and hearing function.