The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

The sniff is part of the olfactory percept

In this review, we use data obtained primarily from humans to argue that sniffs are not merely a stimulus carrier but are rather a central component of the olfactory percept. We argue that sniffs 1) are necessary for the olfactory percept, 2) affect odorant intensity perception and identity perception, 3) drive activity in olfactory cortex, 4) are rapidly modulated in an odorant-dependent fashion by a dedicated olfactomotor system, and 5) are sufficient to generate an olfactory percept of some sort even in the absence of odor.     

Predicting Odor Pleasantness with an Electronic Nose

A primary goal for artificial nose (eNose) technology is to report perceptual qualities of novel odors. Currently, however, eNoses primarily detect and discriminate between odorants they previously “learned”. We tuned an eNose to human odor pleasantness estimates. We then used the eNose to predict the pleasantness of novel odorants, and tested these predictions in naïve subjects who had not participated in the tuning procedure. We found that our apparatus generated odorant pleasantness ratings with above 80% similarity to average human ratings, and with above 90% accuracy at discriminating between categorically pleasant or unpleasant odorants. Similar results were obtained in two cultures, native Israeli and native Ethiopian, without retuning of the apparatus. These findings suggest that unlike in vision and audition, in olfaction there is a systematic predictable link between stimulus structure and stimulus pleasantness. This goes in contrast to the popular notion that odorant pleasantness is completely subjective, and may provide a new method for odor screening and environmental monitoring, as well as a critical building block for digital transmission of smell.     

Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65

Protein phosphorylation ensures the accurate and controlled expression of the genome, for instance by regulating the activities of pre-mRNA splicing factors. Here we report that splicing factor 1 (SF1), which is involved in an early step of intronic sequence recognition, is highly phosphorylated in mammalian cells on two serines within an SPSP motif at the junction between its U2AF65 and RNA binding domains. We show that SF1 interacts in vitro with the protein kinase KIS, which possesses a 'U2AF homology motif' (UHM) domain. The UHM domain of KIS is required for KIS and SF1 to interact, and for KIS to efficiently phosphorylate SF1 on the SPSP motif. Importantly, SPSP phosphorylation by KIS increases binding of SF1 to U2AF65, and enhances formation of the ternary SF1-U2AF65-RNA complex. These results further suggest that this phosphorylation event has an important role for the function of SF1, and possibly for the structural rearrangements associated with spliceosome assembly and function.     

The role of modern biology and medicine in drug development in academia and industry

This symposium addresses careers in drug development in industry; the performance of translational research by academia, industry, and both; and numerous factors pertinent to alliances essential to drug discovery and development. Drug development is a complex process that regularly involves effective collaborations between academic and physician scientists and industry. There are specific occupational factors affecting recruitment of scientists and physicians in drug development programs in industry; ideal backgrounds for successful applicants for positions in industry in drug development; ethical and regulatory considerations particularly germane to the performance of scientists and physicians in drug development programs in industry and at universities; and particular gratifications available to scientists in industry working on drug development. Both similarities and differences characterize the performance of translational research in industry compared with academia. In industry, logistic, operational, and scientific oversight is complex, especially because it often involves relationships with clinical enterprises outside of the corporation. The process is long and arduous from formulation of a good idea in discovery to acceptance of a novel drug in the marketplace. Collaborations and partnerships by industry often involving academia and confrontation of multiple issues are pivotal.     

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.     

Pathways of D-fructose transport in Arthrobacter pyridinolis

Previous work indicated that Arthrobacter pyridinolis can transport d-fructose by either a phosphoenolpyruvate: d-fructose phosphotransferase system or by a respiration-coupled system. The respiration-coupled transport system for d-fructose, which is stimulated by the addition of l-malate, has been characterized in membrane vesicles from d-fructose-grown cells. Such vesicles carry out malate-dependent uptake of d-fructose but not of d-glucose or l-rhamnose, indicating that there is a sugar-specific component to the respiration-coupled transport system. A mutant which is deficient in the d-fructose-specific component was isolated. Vesicles from fructose-glutamate-grown cells of a phosphotransferase-negative strain (AP100) exhibited malate-dependent d-fructose uptake, while phosphoenolpyruvate-dependent uptake was reduced to a small fraction of that seen with vesicles from wild-type cells. Inhibitors of electron transport, carbonyl cyanide m-chlorophenyl hydrazone, 2,4-dinitrophenol and N-ethylmaleimide caused marked inhibition of malate-dependent d-fructose uptake while exerting little or no effect on phosphoenolpyru-vate-dependent transport of the sugar in vesicles from wild-type cells. Activity of a flavin adenine dinucleotide-linked l-malic dehydrogenase was detected in membrane vesicles as well as in whole cells.     

The immunopathology of chronic experimental allergic encephalomyelitis induced in rabbits with bovine proteolipid protein

The role of myelin proteolipid apoprotein (PLP) in the central nervous system (CNS) immune response of rabbits has been investigated by analyzing the immunopathology of chronic experimental allergic encephalomyelitis (EAE) induced by sensitization with PLP. Clinical disease occurred in seven out of nine rabbits sensitized with bovine PLP and monitored for up to 6 mo. Positive delayed hypersensitivity skin test reactions to PLP occurred in all but one of the PLP-sensitized animals. All PLP-sensitized animals had meningeal and CNS parenchymal inflammation that correlated with disease severity. Serial blood samples were stained with a panel of antibodies to rabbit T and B cells, as well as Ia, and large and small mononuclear cell populations were analyzed by flow cytometry. Peripheral leukocyte population staining did not correlate with clinical signs or sensitization to PLP. Cryostat CNS tissue sections were stained with the same set of antibodies by using an immunoperoxidase technique, and positive cells and vessels were counted. T cells and macrophages were numerous and in equal numbers in perivascular parenchymal inflammatory infiltrates, whereas B cells were less numerous (p less than 0.001). T cells also diffusely infiltrated the parenchyma. Most perivascular inflammatory cells and many scattered parenchymal cells were Ia+; Ia vascular expression was increased over controls (p less than 0.001), and also correlated with disease severity. The immunopathology of this chronic EAE model is the same as that of whole CNS tissue- and myelin basic protein-induced EAE in other species, and is similar to that of multiple sclerosis. Cellular immune responses to PLP may therefore contribute to systemic and in situ responses in CNS tissue demyelinating diseases.