The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Glycoside hydrolase family GH85 is a family of endo-β-N-acetylglucosaminidases that is responsible for the hydrolysis of β-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 Å resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (β/α)₈ catalytic domain occurs with two ancillary β-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.     

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.     

Gregor Mendel's classic paper and the nature of science in genetics courses

The discoveries of Gregor Mendel, as described by Mendel in his 1866 paper Versuche uber Pflanzen-Hybriden (Experiments on plant hybrids), can be used in undergraduate genetics and biology courses to engage students about specific nature of science characteristics and their relationship to four of his major contributions to genetics. The use of primary source literature as an instructional tool to enhance genetics students' understanding of the nature of science helps students more clearly understand how scientists work and how the science of genetics has evolved as a discipline. We offer a historical background of how the nature of science developed as a concept and show how Mendel's investigations of heredity can enrich biology and genetics courses by exemplifying the nature of science.     

The neuroleptic chlorpromazine inhibits the cationic and stimulates the anionic phospholipid precursor synthesis in human lymphocytes

The widely used neuroleptic drug chlorpromazine (CPZ) influences membrane functions at the levels of ionic channels and receptors as shown. Here we show the effect of short term treatments by CPZ (30 microM), on the nucleotide-containing phospholipid precursors in human lymphocyte primary cultures. During 60 minutes incubation of the cells, the CDP-ethanolamine (CDP-EA) content was only slightly reduced (87 to 76 pmol/10(6) cells), the amount of CDP-choline (CDP-Ch) was inhibited totally (from 25 to 0 pmol) upon the treatment with 30 microM CPZ under the same conditions. It has been shown earlier, that dCTP can be used as well as CTP for biosynthesis of phospholipids. Thus, the separation of the corresponding ribo- and deoxyribo-liponucleotides was developed. CPZ almost completely inhibited the synthesis of both dCDP-EA and dCDP-Ch under the same conditions The synthesis of the activated liponucleotide precursors, can be measured by incorporation of extracellular 14C-dCyt into both dCDP-EA and dCDP-Ch, as shown earlier. While the cationic deoxyribo-liponucleotide content (dCDP-Ch, dCDP-EA) was decreased, the labelling of the anionic phospholipid precursor dCDP-diacylglycerol (dCDP-DAG) was enhanced several times, it could be labelled only in the presence of CPZ from 14C-dCyd. Thus, a principal disturbance of the membrane phospholipid synthesis is presented (i.e., inhibition of the cationic and enhancement of the anionic dCDP-DAG synthesis). This profound influence on the membrane phospholipids by chlorpromazine, might be the primary effect that contributes to the wide spectrum of CPZ effects on neuronal cells.     

Synthesis of fluorescence-labelled disaccharide substrates of glucosidase II

The fluorescence-labelled disaccharides Glcalpha(1-->3)GlcalphaOR and Glcalpha(1-->3)ManalphaOR, both substrates for the glycoprotein-processing enzyme glucosidase II, were synthesised via the use of a n-pentenyl-derived linker at the anomeric position. This allowed incorporation of a pyrenebutyric acid label, via a sequence of oxidative hydroboration, mesylation, azide displacement, reduction with concomitant global deprotection, and peptide coupling. Selective activation of a fully armed thioglycoside in the presence of n-pentenyl glycosides was readily achieved by the use of methyl triflate as promoter.     

A multicriteria approach to reserve selection: addressing long-term biodiversity maintenance

Existing complementarity-based reserve selection techniquesconcerned with maximal biodiversity representation within minimum landarea do not necessarily ensure the long-term maintenance ofbiodiversity. These approaches often ignore the maintenance of naturalprocesses, turnover of feature diversity and the need to minimisethreats within conservation areas. We address these three emergentissues in the identification of potential avian conservation areas inthe Northern Province of South Africa, by combining ordination andspatial autocorrelation analyses, as well as land transformation datainto complementarity-based reserve selection techniques. Existingconservation areas are biased and inefficient and complementarity-basedmethods do little to correct this skew. The inclusion of speciesassemblage structure as well as the underlying environmental gradientsensures a conservation area network that strives to maintain bothbiodiversity pattern and process. Spatial autocorrelation analysisallows for the identification of areas with high diversity,important areas for the long-term maintenance of biodiversity. Theinclusion of land transformation data leads to viable conservation areanetworks and highlights areas of potential conflict between biodiversityconservation interests and human land-use issues. These combinedimprovements on complementarity-based reserve selection techniques bringus a step closer to ensuring the long-term maintenance of biodiversitywithin conservation areas in the northern province.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Ecological correlates of interindividual distance in the St. Kitts veret (Cercopithecus aethiops sabaeus)

Field data were collected on a free ranging population of vervet monkeys (Cercopithecus aethiops sabaeus) on St. Kitts to test four hypotheses relating cover, risk of predation, and food density to interindividual distance. The results indicated that when food was not a factor, interindividual distance was positively related to the amount of cover in the immediate environment, and therefore to risk of predation. When cover was held constant, distance was inversely related to food density. When the minimum distance for optimal foraging was greater than that required for safety, a compromise distance intermediate between the two predicted values was observed. Cover and food density also predicted the inverse relationship found between age-sex class and interindividual distance. Implications of the above in relation to interindividual competition are discussed.