Enhancing electron affinity and tuning band gap in donor-acceptor organic semiconductors by benzothiadiazole directed C-H borylation.

Electrophilic borylation using BCl3 and benzothiadiazole to direct the C-H functionalisation of an adjacent aromatic unit produces fused boracyclic materials with minimally changed HOMO energy levels but significantly reduced LUMO energy levels. In situ alkylation and arylation at boron using Al(alkyl)3 or Zn(aryl)2 is facile and affords boracycles that possess excellent stability towards protic solvents, including water, and display large bathochromic shifts leading to far red/NIR emission in the solid state with quantum yields of up to 34%. Solution fabricated OLEDs with far red/NIR electroluminescence are reported with EQEs > 0.4%.

Functional role of beta 1 integrin-mediated signalling in the human hair follicle.

Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, beta1 integrins and their ligands are of particular interest. It is not yet known whether beta1 integrins play any role in the regulation of human hair growth and the expression pattern of beta1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for beta1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of beta1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, beta1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two beta1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that beta1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of beta1 integrin-mediated signalling is a suitable candidate for human hair loss management.

Tyrosine phosphorylation regulates nuclear translocation of PKCdelta.

PKCdelta is essential for apoptosis, but regulation of the proapoptotic function of this ubiquitous kinase is not well understood. Nuclear translocation of PKCdelta is necessary and sufficient to induce apoptosis and is mediated via a C-terminal bipartite nuclear localization sequence. However, PKCdelta is found predominantly in the cytoplasm of nonapoptotic cells, and the apoptotic signal that activates its nuclear translocation is not known. We show that in salivary epithelial cells, phosphorylation at specific tyrosine residues in the N-terminal regulatory domain directs PKCdelta to the nucleus where it induces apoptosis. Analysis of each tyrosine residue in PKCdelta by site-directed mutagenesis identified two residues, Y64 and Y155, as essential for nuclear translocation. Suppression of apoptosis correlated with suppressed nuclear localization of the Y --> F mutant proteins. Moreover, a phosphomimetic PKCdelta Y64D/Y155D mutant accumulated in the nucleus in the absence of an apoptotic signal. Forced nuclear accumulation of PKCdelta-Y64F and Y155F mutant proteins, by attachment of an SV40 nuclear localization sequence, fully reconstituted their ability to induce apoptosis, indicating that tyrosine phosphorylation per se is not required for apoptosis, but for targeting PKCdelta to the nucleus. We propose that phosphorylation/dephosphorylation of PKCdelta in the regulatory domain functions as a switch to promote cell survival or cell death.

Mechanisms of integration of cells and extracellular matrices by integrins.

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.

Monoclonal antibodies as probes of integrin priming and activation.

Integrins are a family of heterodimeric, transmembrane receptors that mediate a range of cell-cell and cell-extracellular matrix interactions in an array of physiological and pathophysiological situations. Integrin-mediated cell adhesion is dynamically regulated in vivo to facilitate cell anchorage and movement, but prevents aberrant trafficking and aggregation. Following ligand engagement, integrin signalling imposes a spatial constraint on the assembly of signalling complexes and controls the transduction of mechanical force to the cytoskeleton. This transmembrane passage of signals via integrins is achieved both by clustering of receptors, which makes the ligand and effector engagement more favourable kinetically, and by induction of conformational changes, that theoretically creates ligand and effector binding sites de novo. Clustering and conformational changes can be triggered both from the inside of the cell (resulting in acquisition of ligand-competent conformers) and from the outside (ligand-induced signalling). In this paper, these processes will be described and distinguished by the terms priming and activation, respectively. Although both clustering and conformation are important for integrin function, the latter will be the main focus of this article; in particular, the importance of monoclonal antibodies for the study of integrin shape changes.

Cell adhesion assays.

Cell adhesion makes an important contribution to the maintenance of tissue structure, the promotion of cell migration, and the transduction of information about the cell microenvironment across the plasma membrane. An ability to quantitate adhesion has proven to be extremely valuable for those researchers studying the molecular mechanisms underlying these processes. This article will outline in detail two standard assays used for quantitating the adhesion of cells to an immobilized substrate. First, an attachment assay, which employs a colorimetric detection of bound cells, and second, a spreading assay, which employs phase contrast microscopy to measure the flattening of adherent cells.

DrhoGEF3 encodes a new Drosophila DH domain protein that exhibits a highly dynamic embryonic expression pattern.

The Rho GTPases regulate many different cellular and developmental processes, and activation of Rho GTPase signalling is mediated through interaction with the Dbl homology (DH) protein domain. We describe the expression pattern of DrhoGEF3 (cytological position 61B1-B3), which encodes a new member of the DH domain protein family from Drosophila and is a homologue of the human protein hPEM-2. During gastrulation and germ band extension, DrhoGEF3 exhibits a segmented expression pattern. DrhoGEF3 is subsequently expressed in the visceral mesoderm, at the sites of muscle attachment and in specific groups of sub-epidermal cells. The possible function of such a dynamically expressed signalling molecule is discussed.

Generation of a minimal alpha5beta1 integrin-Fc fragment.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells.

We report here that the novel protein kinase C isoform, PKCdelta, is required at or prior to the level of the mitochondria for apoptosis induced by a diverse group of cell toxins. We have used adenoviral expression of a kinase-dead (KD) mutant of PKCdelta to explore the requirement for PKCdelta in the mitochondrial-dependent apoptotic pathway. Expression of PKCdeltaKD, but not PKCalphaKD, in salivary epithelial cells resulted in a dose-dependent inhibition of apoptosis induced by etoposide, UV-irradiation, brefeldin A, and paclitaxel. DNA fragmentation was blocked up to 71% in parotid C5 cells infected with the PKCdeltaKD adenovirus, whereas caspase-3 activity was inhibited up to 65%. The activation of caspase-9-like proteases by all agents was also inhibited in parotid C5 cells expressing PKCdeltaKD. The ability of PKCdeltaKD to block the loss of mitochondrial membrane potential was similarly determined. Expression of PKCdeltaKD blocked the decrease in mitochondrial membrane potential observed in cells treated with etoposide, UV, brefeldin A, or paclitaxel in a dose-dependent manner. In contrast to the protective function of PKCdeltaKD, expression of PKCdeltaWT resulted in a potent induction of apoptosis, which could be inhibited by co-infection with PKCdeltaKD. These results suggest that PKCdelta is a common intermediate in mitochondrial-dependent apoptosis in salivary epithelial cells.

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

The nature of the active species in bis(imino)pyridyl cobalt ethylene polymerisation catalysts.

Studies on cobalt ethylene polymerisation catalysts bearing bis(imino)pyridine ligands strongly indicate that the activated species is not the anticipated cobalt(II) alkyl cation.

Cell-substrate adhesion assays.

In the intact organism, cells adhere to a variety of substrates. It is possible to investigate the nature of the molecules to which cells adhere in two different assays. In the first protocol presented in this unit, physical adhesion of a cell is assessed by determining the extent to which the cell spreads on a defined substrate--the plate is coated with the test substance, cells are added and allowed time to attach and spread, and the extent of spreading is assessed using phase contrast microscopy. In a second assay, an aliquot of cells is added to the well of a microtiter plate coated with a test adhesion molecule and the cells are allowed to attach. Nonattached cells are removed, and the percent of added cells attached to the substrate is quantified by crystal violet staining.

Integrin structure.

The integrins are a family of alpha,beta heterodimeric receptors that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin cytoskeleton. Integrins are expressed by all multicellular animals, but their diversity varies widely among species; for example, in mammals, 19 alpha and 8 beta subunit genes encode polypeptides that combine to form 25 different receptors, whereas the Drosophila and Caenorhabditis genomes encode only five and two integrin alpha subunits respectively. Thousands of studies over the last two decades have investigated the molecular, cellular and organismal basis of integrin function. Gene deletion has demonstrated essential roles for almost all integrins, with the defects suggesting widespread contributions to both the maintenance of tissue integrity and the promotion of cellular migration. Integrin-ligand interactions are now considered to provide physical support for cells in order to maintain cohesion, to permit the generation of traction forces to enable movement, and to organize signalling complexes to modulate differentiation and cell fate. Animal-model studies have also shown that integrins contribute to the progression of many common diseases, and have implicated them as potential therapeutic targets. The use of anti-integrin monoclonal antibodies and ligand-mimetic peptides has validated this suggestion for inflammatory, neoplastic, traumatic and infectious conditions. Thus, to understand more about the mechanisms underlying tissue organization and cellular trafficking, and to identify approaches for regulating these processes in disease, there is intense interest in determining the molecular basis of integrin function. It is important to state at the outset that the tertiary structure of the integrin dimer is unknown. Our current understanding of the molecular basis of integrin function is therefore compiled from the results of a large number of studies that have employed a wide range of complementary technologies.

Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha5beta1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding.

Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, alphavbeta3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin alpha5beta1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to alpha5beta1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins alpha5beta1 and alphavbeta3 and evidence is provided that alpha5beta1 might be an important FMDV receptor in vivo.

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit.

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).