Three-dimensional nanothermometry below the diffraction limit.

Lanthanide-doped nanothermometers are used to measure temperature through changes in their emission characteristic with sensitivities of up to a few %/K. In contrast to their sensitivity, their spatial resolution, which is of critical importance for various applications, has not been thoroughly studied and optimized. We numerically investigated the improvement in spatial resolution of nanothermometers with a stimulated emission depletion microscopy approach. Fundamental relationships between spatial and temperature resolution were identified by using different beam parameters for the excitation and depletion beams. Our simulations predict contactless temperature measurement below the diffraction limit with temperature resolution of ±1.25K. We further studied the influence of sample thickness and position on both temperature and spatial resolution and showed the potential of three-dimensional measurements.

Failure to engage the public in issues related to inland fishes and fisheries: strategies for building public and political will to promote meaningful conservation.

Generating awareness of environmental conservation issues among the public is essential if there is an expectation of them to alter their behaviour, facilitate informed decisions and engage governments or regulatory authorities to take action. There are, however, exceedingly few public engagement success stories related to inland fishes and fisheries policy and resource allocation decisions. Inland aquatic resources and their associated fisheries provide employment, recreation, culture and, in developing regions, a considerable proportion of human nutrition and food security. Freshwater fishes are incredibly diverse but are among the most endangered organisms globally. Many threats to inland fisheries are driven largely by externalities to inland fisheries. The purpose of this paper is to draw attention to the role and plight of inland fishes and fisheries, and the need to generate the public and political will necessary to promote meaningful conservation. With this paper, the extent to which the scientific and environmental management communities have failed to engage the public in issues related to inland fishes and fisheries is characterized. Next, the barriers or factors that serve as the basis for the problem with public engagement are identified. The paper concludes by identifying strategies, including those focused on environmental education initiatives, for building the public and political will necessary to promote meaningful conservation of inland fishes and fisheries in developed and developing countries. Scientists, environmental managers, non-governmental organizations, politicians, regulatory authorities and the media all have important roles to play in overcoming challenges to inland fisheries. Failure to engage the public in freshwater conservation and management issues will impede efforts to stem the loss of freshwater habitats, fisheries and aquatic biodiversity. Thankfully, there are opportunities to learn from success stories related to other environmental issues and initiatives that have been successful in marine fish conservation.

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides: part III: pH-sensitive headgroups.

As part of a series of papers, the influence of carbohydrate headgroups and aliphatic chains on the mesogenic properties of glycolipids was investigated. Alkyl glycosides with different types of aliphatic chains were synthesised. Neutral glycolipids were oxidized to their uronic acid derivatives, using the well established TEMPO-oxidation. For comparison a 6-deoxy-6-amino alkylglucopyranoside was synthesised. In addition, the thermotropic and lyotropic phase behaviour of the synthesised compounds were investigated. The thermotropism was characterised by polarising microscopy, the lyotropism by the contact preparation method.

Syntheses of imido-substituted glycosans and their photocyclisation towards highly functionalised heterotricycles.

Reaction of O-protected amino-1,6-anhydro-beta-D-hexopyranoses with succinic or glutaric anhydride and subsequent intramolecular acylation afforded the succinimido- and glutarimido-substituted glycosans. Irradiation with UV light of 254 nm wavelength led to gamma-hydrogen abstraction at the pyranose ring by the excited carbonyl function. The stereoselective recombination of the resulting 1,4-diradicals gave annelated azetidinols, which fragmented by a retrotransannular ring opening reaction to give the glycosan-annelated azepanedione and azocanedione systems, respectively.

The first bovine beta 1,4-galactosyltransferase reaction with an acyclic acceptor substrate, 3-acetamido-1,2-propanediol, to yield a 3-O-beta-D-galactopyranosyl-sn-glycerol skeleton.

[figure: see text] Reactivity of bovine beta 1,4-galactosyltransferase was examined for a series of acyclic acceptor substrates both in the presence and the absence of alpha-lactalbumin (alpha-La). It was found that this enzyme could utilize (R)-3-acetamido-1,2-propanediol (1) as an acceptor substrate regardless of the cofactor protein. The product was determined to be 1-O-beta-D-galactopyranosyl-(R)-3-acetamido-1,2-propanediol (2). Glycerol without the acetamido group was inactive, indicating that this functional group plays a key role in the enzyme reaction.

Chemoenzymatic synthesis of sialyl oligosaccharides with sialidases employing transglycosylation methodology.

A series of sialyloligosaccharides was synthesized using the transglycolytic activity of the sialidases from Vibrio cholerae, Clostridium perfringens, Salmonella typhimurium, and Newcastle disease virus. According to their hydrolytic activities the sialidases from V. cholerae and C. perfringens catalyze preferentially the formation of sialyl alpha(2-6)-linkages whereas the sialidases from S.typhimurium and Newcastle disease virus show a distinct preference for alpha(2-3) directed sialylations. Using combined chemical and enzymatic methodologies structures such as T-(Thomsen-Friedenreich) antigen [beta-D-Gal-(1-3)-alpha-D-GalNAc-OThr], Tn-(Thomsen nouveau) antigen (alpha-D-GalNAc-OThr) and beta-D-Gal-(1-4)-alpha-D-2-deoxy-Gal-OMe were sialylated in alpha(2-3)- and alpha(2-6)-positions regioselectively or in high regioisomeric excess and purified by simple isolation procedures. Depending on the enzyme source and acceptor structure yields for transsialylation varied between 10 and 30%.

Enzymatic synthesis of fucose-containing disaccharides employing the partially purified alpha-L-fucosidase from Penicillium multicolor.

The alpha-L-Fucp-(1 --> 3)-D-GlcpNAc disaccharide structure is a vital core unit of the oligosaccharide components of glycoconjugates isolated from human milk and blood group substances. Alpha-L-Fucosidase from Penicillium multicolor catalyses the transfer of L-fucose from donor structures such as alpha-L-FucpOpNP and alpha-L-FucpF to various GlcpNAc derivatives and Glcp, forming alpha-(1 --> 3) linkages. The synthesis of several biologically relevant disaccharides including alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOMe, alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-beta-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-D-GlcpNAc and alpha-L-Fucp-(1 --> 3)-D-Glcp has been achieved in up to 34% yields by application of this enzyme.

Synthesis of novel donor mimetics of UDP-Gal, UDP-GlcNAc, and UDP-GalNAc as potential transferase inhibitors.

For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.

Galactan biosynthesis in snails: a comparative study of beta-(1--> 6) galactosyltransferases from Helix pomatia and Biomphalaria glabrata.

Adult snails synthesize in their albumen glands a polysaccharide which is composed exclusively of D- or D- and L-galactose (Gal) residues which are interglycosidically linked by 1 --> 3 and 1 --> 6 bonds. It is the only carbohydrate source for embryos and freshly hatched snails. Two galactosyltransferases are described in this study which are most likely involved in the biosynthesis of this polysaccharide. One identified in Helix pomatia acts on oligosaccharides and could be used to synthesize a tetrasaccharide when the branched trisaccharide D-Gal-beta-(1 --> 3)-[D-Galbeta-(1 --> 6)]-D-Galbeta-1 --> OMe was offered as acceptor. This enzyme, requiring Mg++-and Mn++-ions for activity, introduced a linear beta-(1 --> 6) linkage at the terminal non-reducing ends and was not detected in Biomphalaria glabrata. The other enzyme, which introduced beta-(1 --> 6) linkages at subterminal D-Gal residues, thus forming branching points in the polysaccharide, was found in H. pomatia, Arianta arbustorum and B. glabrata with comparable activities. With the enzyme preparation of H. pomatia, up to four D-Gal residues were introduced into vicinal positions, forming single-membered side chains, if a hexasaccharide with five linearly beta-(1 --> 3)-linked D-Gal residues was offered as a acceptor. The multiple-branched structure formed is typical for snail galactans, making this enzyme a prime candidate for the branching enzyme in galactan synthesis. The enzyme activity could be solubilized and purified by affinity chromatography. In SDS-polyacrylamide electrophoresis, the Helix-derived eluate displayed two bands (68, 37 kDa) and that of Biomphalaria five bands (68, 63, 17.5; 15; 13 kDa). The purified material showed only 8% of the total activity of the crude extracts, but it could be shown that a phosphatase present in the crude extract can degrade UDP formed in the transfer reaction and thus drive the reaction to completion.

Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins.

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and alpha 2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.

Hydrolase-catalyzed beta-mannosylations.

Transmannosylations catalysed by beta-mannosidase from snail viscera or beta-galactosidase from Aspergillus oryzae were accomplished with 4-nitrophenyl beta-D-mannopyranoside as donor substrate. With suitable hydrophobic acceptor molecules preferentially beta1-4-linked disaccharides were obtained. The activities of both glycosidases in buffer cosolvent mixtures were determined, and conditions for their immobilization were elaborated and optimized. A model of the enzymic transfer mechanism is suggested.

Preparation of modified glycosyl glycerol derivatives by glycal rearrangement.

By Lewis acid catalysed allylic rearrangement reactions of various glycals with glycerol derivatives 2,3-unsaturated mono- and disaccharide glycosyl glycerol derivatives were obtained in good yields. A triglycosyl glycerol was successfully synthesized in a straightforward three step way from 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol.

In vitro alpha1-3 or alpha1-4 fucosylation of type I and II oligosaccharides with secreted forms of recombinant human fucosyltransferases III and VI.

Transgalactosylation of chitobiose and chitotriose employing beta-galactosidase from bovine testes yielded mixtures with beta1-3 linked galactose (type I) and beta1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human alpha1,3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewis(a) structure, whereas approximately 10% fucosylation of the type II isomers to the Lewis(x) oligosaccharides was observed in long-term incubations. Employing large amounts of human alpha1,3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewis(x) product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely alpha1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a beta1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an alpha1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewis(a) oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.

Synthesis of an N-glucoasparagine analog as a building block for a V3-loop glycopeptide from gp120 of HIV-I.

The preparative synthesis of a new N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine mimetic 1, starting from 2-amino-1,5-anhydro-2-deoxy-glucitol hydrochloride and Z-Asp-(OH)-OBn is described. This glycosyl-amino acid unit 1 is expected to show higher stabilities towards in vivo conditions. Further, the use of 1 as building block for the synthesis of modified glycopeptides using solid phase support is reported. The glycopeptide Ac-SXNTRKSIHIGPGRAF-NH2 having sugar-modified Asn2 mimics parts of the V3-loop structure containing the principle neutralizing determinant (PND) of HIV-1 and the naturally conserved glycosylation site within the V3 loop.

NKR-P1A protein, an activating receptor of rat natural killer cells, binds to the chitobiose core of uncompletely glycosylated N-linked glycans, and to linear chitooligomers.

NKR-P1 represent a family of activating receptors in rodent natural killer cells related to C-type animal lectins. We identify here the elements involved in the reactivity of the major receptor of rat, NKR-P1A, with N-linked oligosaccharides of glycoproteins. Plate inhibition assays with isolated, structurally defined N-glycans as inhibitors of binding of NKR-P1A to GlcNAc16-BSA revealed that the removal of both the external sialic acids and the penultimate galactose residues resulted in attaining of significant inhibitory activities. Surprisingly, additional plate inhibition and glycoprotein overlay experiments brought evidence that the core chitobiose, depending on its substitution, can per se support the interaction with NKR-P1A. In a series of linear chitooligomers (n = 2-7), the inhibitory activities reached a maximum for the chitotetraose. The ability of NKR-P1 to recognize both the periphery and the core region of complex type oligosaccharides may define its dual specificity towards carbohydrate components of eukaryotic (e.g., tumor) cell surfaces, but also reflect an evolutionarily conserved reactivity with microbial saccharides important in immune recognition and signaling functions.

Galactosylation with beta-galactosidase from bovine testes employing modified acceptor substrates.

In this study beta1-3 linked analogues of the T-antigen determinant were synthesized in preparative scale by transgalactosylation using beta-galactosidase from bovine testes to give synthetic antigens. Acceptors with modifications of the sugar residue such as alpha-glycosylated spacers, as well as GlcNAc-alphaOR- and 2dGal-alphaOR-substrates opened further possibilities for galactosylation.

Specificity of amylases and cyclodextrin-glucanotransferase in reactions with 2-deoxy-maltooligosaccharides.

2-Deoxy-maltooligosaccharides of different chain length were tested as substrates for exo- and endo-amylases. Cleavage occurred with beta-amylase, yielding 2,2'-dideoxy-maltose, and with amyloglucosidase. With the alpha-amylase from Thermomonospora curvata tris-(2-deoxy)-maltotriose and the corresponding tetra- and pentasaccharides were formed. Porcine pancreatic alpha-amylase did not tolerate the deoxygenated substrate, nor were cyclization experiments with cyclodextrin-glucanotransferase (CGT) successful. In a coupling reaction with CGT, however, a series of transfer products to the acceptor 2-deoxyglucose were obtained.

Further syntheses employing phosphorylase.

Maltopentaose was immobilized on silica gel and used as a recyclable primer in the reaction of glycogen phosphorylase with D-glucal. An improved method to obtain 2-deoxy-alpha-D-arabino-hexopyranosyl phosphate (4) by using this catalyst as well as the specific synthesis of low molecular weight, water-soluble 2-deoxy-maltooligosaccharides (12, 13, 14 and 15) are described. Further investigations with modified phosphorylase substrates showed that mannosyl phosphate (16) can be slowly transferred to the primer maltotetraose. alpha-1,4-Mannosyl-maltotetraose (17) and its degradation product alpha-1,4-mannosyl-maltose (18) were identified.