Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification

Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP-glucose:flavonoid 3-O-glycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health-promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three-dimensional structure of VvGT1 has also been determined, both in its 'Michaelis' complex with a UDP-glucose-derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.     

Hydrostatic Pressure and Temperature Effects on the Membranes of a Seasonally Migrating Marine Copepod

Marine planktonic copepods of the order Calanoida are central to the ecology and productivity of high latitude ecosystems, representing the interface between primary producers and fish. These animals typically undertake a seasonal vertical migration into the deep sea, where they remain dormant for periods of between three and nine months. Descending copepods are subject to low temperatures and increased hydrostatic pressures. Nothing is known about how these organisms adapt their membranes to these environmental stressors. We collected copepods (Calanoides acutus) from the Southern Ocean at depth horizons ranging from surface waters down to 1000 m. Temperature and/or pressure both had significant, additive effects on the overall composition of the membrane phospholipid fatty acids (PLFAs) in C. acutus. The most prominent constituent of the PLFAs, the polyunsaturated fatty acid docosahexanoic acid [DHA – 22:6(n-3)], was affected by a significant interaction between temperature and pressure. This moiety increased with pressure, with the rate of increase being greater at colder temperatures. We suggest that DHA is key to the physiological adaptations of vertically migrating zooplankton, most likely because the biophysical properties of this compound are suited to maintaining membrane order in the cold, high pressure conditions that persist in the deep sea. As copepods cannot synthesise DHA and do not feed during dormancy, sufficient DHA must be accumulated through ingestion before migration is initiated. Climate-driven changes in the timing and abundance of the flagellated microplankton that supply DHA to copepods have major implications for the capacity of these animals to undertake their seasonal life cycle successfully.     

Spatial distributions of Southern Ocean mesozooplankton communities have been resilient to long‐term surface warming

The biogeographic response of oceanic planktonic communities to climatic change has a large influence on the future stability of marine food webs and the functioning of global biogeochemical cycles. Temperature plays a pivotal role in determining the distribution of these communities and ocean warming has the potential to cause major distributional shifts, particularly in polar regions where the thermal envelope is narrow. We considered the impact of long‐term ocean warming on the spatial distribution of Southern Ocean mesozooplankton communities through examining plankton abundance in relation to sea surface temperature between two distinct periods, separated by around 60 years. Analyses considered 16 dominant mesozooplankton taxa (in terms of biomass and abundance) in the southwest Atlantic sector of the Southern Ocean, from net samples and in situ temperature records collected during the Discovery Investigations (1926–1938) and contemporary campaigns (1996–2013). Sea surface temperature was found to have increased significantly by 0.74°C between the two eras. The corresponding sea surface temperature at which community abundance peaked was also significantly higher in contemporary times, by 0.98°C. Spatial projections indicated that the geographical location of community peak abundance had remained the same between the two eras despite the poleward advance of sea surface isotherms. If the community had remained within the same thermal envelope as in the 1920s–1930s, community peak abundance would be 500 km further south in the contemporary era. Studies in the northern hemisphere have found that dominant taxa, such as calanoid copepods, have conserved their thermal niches and tracked surface isotherms polewards. The fact that this has not occurred in the Southern Ocean suggests that other selective pressures, particularly food availability and the properties of underlying water masses, place greater constraints on spatial distributions in this region. It further demonstrates that this community is thermally resilient to present levels of sea surface warming.     

Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols

1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight chain spacer-arms by a set of simple standard procedures. The spacer-arms' terminal functional groups, primary amines, were employed to introduce fluorescent tags such as dansyl and dapoxyl moieties. Resulting derivatives in the D-xylo, D-gluco, D-galacto as well as GlcNAc series showed distinctly improved glycosidase inhibitory activities compared to parent compounds and are designed to be useful analytical tools.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

Lung tumour induction in mice after uniform and non-uniform external thoracic X-irradiation

The aim of this study was to investigate the validity of the ICRP procedure of using average tissue/organ dose in estimating carcinogenic risk. It has been suggested that highly non-uniform exposure ('hot spots') is much more carcinogenic than an equivalent dose delivered uniformly. In a series of experiments, mice were irradiated with X-rays either uniformly to the thorax or non-uniformly with 72 1-mm microbeams which irradiated approximately 20 per cent of the total lung volume. Two experiments involving uniform irradiation showed a peaked tumour incidence curve with a maximum at 5 Gy. The first 'microbeam' study also produced a pronounced peak in the dose response with a maximum tumour incidence at 1 Gy average lung dose or 5 Gy to the irradiated lung tissue. This implied the use of average tissue dose might underestimate the carcinogenic hazard of non-uniform exposure. Later, more extensive, microbeam experiments failed to replicate this finding. The results were nearly similar to those for uniform irradiation, with a slight increase in tumour incidence from 2.5-5.0 Gy average lung dose. These results imply that for these irradiation conditions the ICRP dose averaging procedure remains valid.