Methods for the synthesis of polyhydroxylated piperidines by diastereoselective dihydroxylation: Exploitation in the two-directional synthesis of aza-C-linked disaccharide derivatives

Background: Many polyhydroxylated piperidines are inhibitors of the oligosaccharide processing enzymes, glycosidases and glycosyltransferases. Aza-C-linked disaccharide mimetics are compounds in which saturated polyhydroxylated nitrogen and oxygen heterocycles are linked by an all-carbon tether. The saturated oxygen heterocycle has the potential to mimic the departing sugar in a glycosidase-catalysed reaction and aza-C-linked disaccharide mimetics may, therefore, be more potent inhibitors of these enzymes. Results: The scope, limitations and diastereoselectivity of the dihydroxylation of stereoisomeric 2-butyl-1-(toluene-4-sulfonyl)-1,2,3,6-tetrahydro-pyridin-3-ols is discussed. In the absence of a 6- substituent on the piperidine ring, the Upjohn (cat. OsO4, NMO, acetone-water) and Donohoe (OsO4, TMEDA, CH2Cl2) conditions allow complementary diastereoselective functionalisation of the alkene of the (2R*,3R*) diastereoisomer. However, in the presence of a 6-substituent, the reaction is largely controlled by steric effects with both reagents. The most synthetically useful protocols were exploited in the two-directional synthesis of aza-C-linked disaccharide analogues. A two-directional oxidative ring expansion was used to prepare bis-enones such as (2R,6S,2'S)-6- methoxy-2-(6-methoxy-3-oxo-3,6-dihydro-2H-pyran-2-ylmethyl)-1-(toluene-4-sulfonyl)-1,6- dihydro-2H-pyridin-3-one from the corresponding difuran. Selective substitution of its N,O acetal was possible. The stereochemical outcome of a two-directional Luche reduction step was different in the two heterocyclic rings, and depended on the conformation of the ring. Finally, twodirectional diastereoselective dihydroxylation yielded seven different aza-C-linked disaccharide analogues. Conclusion: A two-directional approach may be exploited in the synthesis of aza-C-linked disaccharide mimetics. Unlike previous approaches to similar molecules, neither of the heterocyclic rings is directly derived from a sugar, allowing mimetics with unusual configurations to be prepared. The work demonstrates that highly unsymmetrical molecules may be prepared using a two directional approach. The deprotected compounds may have potential as inhibitors of oligosaccharide-processing enzymes and as tools in chemical genetic investigations

Polyglutamine repeat proteins disrupt actin structure in Drosophila photoreceptors.

Expansions of polygutamine-encoding stretches in several genes cause neurodegenerative disorders including Huntington\u27s Disease and Spinocerebellar Ataxia type 3. Expression of the human disease alleles in Drosophila melanogaster neurons recapitulates cellular features of these disorders, and has therefore been used to model the cell biology of these diseases. Here, we show that polyglutamine disease alleles expressed in Drosophila photoreceptors disrupt actin structure at rhabdomeres, as other groups have shown they do in Drosophila and mammalian dendrites. We show this actin regulatory pathway works through the small G protein Rac and the actin nucleating protein Form3. We also find that Form3 has additional functions in photoreceptors, and that loss of Form3 results in the specification of extra photoreceptors in the ey

Adhesion signalling complexes

SummaryIntercellular communication in metazoa not only requires autocrine, paracrine and exocrine signalling systems, but it also relies on the structural and positional information encoded in extracellular matrices (ECMs). Most cells in tissues are structurally and functionally integrated with their surrounding ECM in a highly organised manner involving thousands of dynamic connections. On the intracellular face of these linkages, adhesion receptors — principally integrins and syndecans — link the cytoskeleton to the plasma membrane and compartmentalise cytoplasmic signalling events, whereas at the extracellular face the same receptors direct and organise the deposition of the ECM itself. Adhesion receptors transduce mechanical force bidirectionally across the plasma membrane by tethering variably deformable ECMs to the contractile cytoskeleton (Figure 1), and they translate the topography and composition of the ECM into chemical signals that determine behaviour. The membrane-proximal functions of adhesion receptors in turn trigger distal processes within cells, such as alterations in the direction of cell movement and the regulation of gene transcription, and long-range effects outside cells, such as the construction of ECM networks and consequent shaping of higher-order tissue structure. Given the diverse and fundamental roles attributed to adhesion, it is understandable that adhesion receptor engagement has been reported to alter the flux through virtually all major signalling pathways

Basement membrane ligands initiate distinct signalling networks to direct cell shape

Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913

Global analysis reveals the complexity of the human glomerular extracellular matrix.

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456

Faecal haemoglobin concentration in adenoma, before and after polypectomy, approaches the ideal tumour marker

BACKGROUND: Polypectomy may be performed at colonoscopy and then subsequent surveillance undertaken. It is thought that faecal haemoglobin concentration (f-Hb), estimated by quantitative faecal immunochemical tests (FIT), might be a useful tumour marker. METHODS: Consecutive patients enrolled in colonoscopy surveillance were approached at two hospitals. A specimen for FIT was provided before colonoscopy and, ideally after 3 weeks, a second FIT sample from those who had polypectomy. A single FIT system (OC-Sensor io, Eiken Chemical Co., Ltd) was used to generate f-Hb. RESULTS: 1103 Patients were invited; 643 returned a FIT device (uptake: 58.3%). Four patients had known inflammatory bowel disease (IBD) and were excluded, leaving 639 (57.9%) with an age range of 25–90 years (median 64 years), 54.6% male. Of 593 patients who had a f-Hb result and completed colonoscopy, advanced neoplasia was found in 41 (6.9%); four colorectal cancer (CRC): 0.7% and 37 advanced adenoma (AA): 6.3%, and a further 127 (21.4%) had non-advanced adenoma (NAA). The median f-Hb was significantly greater in AA as compared to NAA; 6.0 versus 1.0 μg Hb/g faeces, p < 0.0001.134/164 (81.7%) of invited patients returned a second FIT device: 28 were patients with AA in whom median pre-polypectomy f-Hb was 19.2, falling to 3.5 μg Hb/g faeces post-polypectomy, p = 0.01, and 106 with NAA had median pre-polypectomy f-Hb 0.8 compared to 1.0 μg Hb/g faeces post-polypectomy, p = 0.96. CONCLUSIONS: Quantitative FIT could provide a good tumour marker in post-polypectomy surveillance, reduce colonoscopy requirements and minimise potential risk to patients