DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Methods for the isolation of viable gonadotropin-releasing hormone (LRF) neurons

Studies were conducted in order to determine if selected neurons could be isolated from the brain using Sepharose-linked recognition complexes directed against or related to the biosynthetic/neurosecretory product of the desired neuronal population. Immunoreactive LRF neurons were precipitated when dispersed cells of adult male rats were incubated successively in media containing free LRF antiserum followed by the exposure of LRF bound to Sepharose-4B. The radioimmunoassayable LRF content of the isolated cells was 88% of that contained in fresh frozen tissue of a contemporary group of rats and trypan blue exclusion indicated that at least 85% of the neurons were viable. Furthermore, based on immunocytochemistry and cresyl violet staining in combination with immunocytochemistry, the isolated cell fraction appeared to be free from other types of cells and also exhibited assayable LRF release when challenged with potassium. These results suggest that the neuroendocrine properties of hypothalamic neurons may be exploited in order to isolate viable cells for acute in vitro experiments.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell–matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.     

Identification,Quantification, and System Analysis of Protein N‐ε Lysine Methylation in Anucleate Blood Platelets

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N‐ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl‐lysine (meK) proteome of anucleate blood platelets is characterized. With high‐resolution, multiplex MS methods, 190 mono‐, di‐, and tri‐meK modifications are identified on 150 different platelet proteins—including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin‐like protein (DBNL, Hip‐55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.     

Visualisation of hyaluronan and hyaluronan-binding proteins within ovine vertebral cartilages using biotinylated aggrecan G1-link complex and biotinylated hyaluronan oligosaccharides

The aim of this study was to localise hyaluronan (HA)-binding proteins (HABPs) in ovine vertebral tissues using biotinylated HA oligosaccharides (bHA oligos) as novel affinity probes and to compare this with the distribution of tissue HA visualised using biotinylated aggrecan G1 domain-link protein complex. The bHA oligos, with a size of 6-18 disaccharides were prepared by partial digestion of HA with ovine testicular hyaluronidase, labelled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaluronan and HABPs were both prominent pericellular components of hypertrophic cells of the vertebral epiphyseal growth plate and enlarged cells in the cartilaginous end plate of the disc. The bHA oligo probe also visualised HABPs intracellularly in hypertrophic cells, which also contained intracellular HA. Monolayer cultures of ovine annulus fibrosus and nucleus pulposus cells rapidly internalised the bHA oligo affinity probe which was subsequently visualised by indirect fluorescence using avidin-FITC, to cytoplasm and discrete nuclear regions. The results indicate that the abundant pericellular and intracellular HA associated with cartilaginous cells in the vertebral tissues is colocalised with HABPs. The bHA oligo affinity probe may have further applications in investigations of intracellular HABPs, HA endocytosis and the roles they play in cellular regulatory processes.     

Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity

Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.     

A spatial analytical approach for selecting reintroduction sites for burbot in English rivers

1. Availability of suitable habitat is a prerequisite for species reintroduction success, and to ensure population persistence, investigations of a species’ habitat utilisation throughout its life history should be conducted as part of a feasibility study. 2. Habitat utilisation models for burbot, Lota lota, developed using data from field studies conducted in France and Germany and information from the literature were used to assess the feasibility of reintroducing burbot into rivers of its former native range in eastern England. 3. Per cent tree roots, aquatic vegetation and flow types were important predictors of adult burbot abundance. Furthermore, the habitat utilisation models were supplemented with information from the literature, which suggested that off‐channel habitat such as wetlands and backwaters is important for spawning and nursery stages. 4. An assessment of the habitat availability in the rivers of the burbot’s former native range using variables related to spawning and nursery and adult life stages showed that although adult habitat was widely distributed, the availability of spawning and nursery habitat was less abundant, potentially limiting successful reestablishment. 5. Potential suitable habitat was concentrated in the central and southern areas of the species’ former English distribution. Overall, rivers of the burbot’s former range potentially afford suitable habitat to sustain a reintroduced population. However, sites should be preferentially selected on the basis of having appropriate spawning and nursery areas.     

Perlecan, the "jack of all trades" proteoglycan of cartilaginous weight-bearing connective tissues

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.