Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

A combination of recombinant FKP and α-(1→3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.     

Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid

7-Fluoro sialic acid was prepared and activated as cytidine monophosphate (CMP) ester. The synthesis started with d-glucose, which was efficiently converted into N-acetyl-4-fluoro-4-deoxy-d-mannosamine. Aldolase catalyzed transformation yielded the corresponding fluorinated sialic acid which was activated as CMP ester using three different synthetases in the presence as well as in the absence of pyrophosphatase which possesses inhibitory properties. Finally, conditions were optimized to perform a one-pot reaction starting from fluorinated mannosamine, which yielded the 7-fluoro-7-deoxy-CMP-sialic acid by incubation with three enzymes.     

A two-step fermentation process for efficient production of penta-N-acetyl-chitopentaose in recombinant Escherichia coli

The nodC gene from Mesorhizobium loti was cloned into E. coli, leading to production of chitin oligosaccharides (COs)—mainly penta-N-acetyl-chitopentaose. A two-step fermentation procedure was then developed which gave 930 mg CO/L with a productivity of 37 mg/l·h.     

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.     

Radioimmunoassay for pmol-quantities of indole-3-acetic acid for use with highly stable [125I]- and [3H]IAA derivatives as radiotracers

A radioimmunoassay for the detection of as little as 0.5–1 pmol indole-3-acetic acid (IAA) in unpurified or partially purified plant extracts is described. The assay makes use of either IAA[¹²⁵I]tyrosine methyl ester or [³H]IAA methyl ester as radioactive antigens and IAA methyl ester as the assay standard (measuring range: 1–200 pmol). Levels of extractable IAA in a number of biological samples have been estimated.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxy acetic acid - DMF dimethyl formamide - GC-MS gas chromatography-mass spectroscopy - IAA indole-3-acetic acid - RIA radioimmunoassay - SICM selected ion current monitoring - TLC thin layer chromatography - TME tyrosine methyl ester Part 18 in the series: Use of immunoassay in plant science     

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Phosphorus-32 bioelimination by arthropod predators fed labeled eggs ofHeliothis virescens [Lep.: Noctuidae]

Adults ofGeocoris punctipes (Say),Tropiconabis capsiformis Germar,Nabis roseipennis Reuter andColeomegilla maculata (DeGeer) were confined inside petri dishes and fed phosphorus-32 (^32p)-labeledHeliothis virescens (F.) eggs. Observations of bioelimination of^32p over a 72 h period allowed derivation of equations for predicting the number ofH. virescens eggs ingested by each species. Twenty-four to 72 h after eggs were eaten, the percentages of^32p eliminated ranged from ca. 14% forG. punctipes to 42 % forT. capsiformis. Cautious use of the results will aid researchers in assessing predation on^32p labeledHeliothis. Publication N^o 5935. Mississippi Agricultural and Forestry Experiment Station, Mississippi State, MS 39762.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Alkylation of [3H]8-OH-DPAT binding sites in rat cerebral cortex and hippocampus

The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or [³H]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus could be inhibited by serotonin (5-HT) and buspirone, and by the 5-HT antagonists propranolol, NAN-190, pindolol, pindobind-5-HT_1A, WAY100135, spiperone and ritanserin. All competition curves, except for ritanserin, best fitted a two-site model. In vitro treatment of the membranes withN-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused dose-dependent decreases of binding; the inhibition curves were biphasic, and the effects irreversible. Reduction of disulfide bonds withl-dithiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reversible in hippocampus. In the latter region, but not in cerebral cortex, previous occupancy by [³H]8-OH-DPAT partially protected binding from the effects of bothL-DTT and NEM, suggesting that the thiol groups in the receptor recognition site(s) of this brain region are readily accessible. The binding characteristics were examined with the aid of saturation curves, carried out with increasing concentrations, up to 140 nM, of [³H]8-OH-DPAT. The saturation data were suggestive of a two-site receptor model incorporating a high-affinity site (Kh of 0.3–0.5 nM) corresponding to the 5-HT_1A receptor, and a low-affinity site (Kl ofca 25 nM). After in vivo alkylations, carried out by treating rats withN-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ), the saturation curves from both control and EEDQ-treated rats were again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrease of 5-HT_1A receptor activity was noted; this loss was greater in hippocampus than in cerebral cortex. Since the decrease in 5-HT_1A receptors was not associated with changes in low-affinity binding, the results suggest independent regulations of the two [³H]8-OH-DPAT binding proteins. Altogether, the present data further supports the notion that [³H]8-OH-DPAT, besides labelling 5-HT_1A receptors, also binds to other structures in rat cerebral cortex and hippocampus. Special issue dedicated to Dr. Kinya Kuriyama     

Chemoenzymatic synthesis and antileukemic activity of novel C9- and C14-functionalized parthenolide analogs

Parthenolide is a naturally occurring terpene with promising anticancer properties, particularly in the context of acute myeloid leukemia (AML). Optimization of this natural product has been challenged by limited opportunities for the late-stage functionalization of this molecule without affecting the pharmacologically important α-methylene-γ-lactone moiety. Here, we report the further development and application of a chemoenzymatic strategy to afford a series of new analogs of parthenolide functionalized at the aliphatic positions C9 and C14. Several of these compounds were determined to be able to kill leukemia cells and patient-derived primary AML specimens with improved activity compared to parthenolide, exhibiting LC₅₀ values in the low micromolar range. These studies demonstrate that different O–H functionalization chemistries can be applied to elaborate the parthenolide scaffold and that modifications at the C9 or C14 position can effectively enhance the antileukemic properties of this natural product. The C9-functionalized analogs 22a and 25b were identified as the most interesting compounds in terms of antileukemic potency and selectivity toward AML versus healthy blood cells.     

Allopregnanolone increase in striatal N-methyl-D-aspartic acid evoked [3H]dopamine release is estrogen and progesterone dependent

1. The neurosteroids are compounds derived from steroid hormones and synthesized in the nervous system. They can modulate different neurotransmitter pathways. In previous work we demonstrated that progesterone modulates dopamine release induced by the glutamatergic agonist N-methyl-D-aspartic acid (NMDA).2. The aim of this work was to evaluate a possible modulatory role of the progesterone metabolite allopregnanolone on NMDA-evoked [³H]dopamine release from corpus striatum slices obtained from cycling and ovariectomized female rats.3. We used a dynamic superfusion method to evaluate the release of [³H]dopamine. Allopregnanolone at 50–600 nM was added to the superfusion buffer (Krebs–Ringer–bicarbonate–glucose, pH 7.4, with constant O₂/CO₂ gassing). The results are expressed as a percentage over basal [³H]dopamine loaded by the tissue.4. Allopregnanolone (50 and 100 nM) increased the NMDA-evoked[³H]dopamine release from estrus rats. The remaining doses did not show significant changes in the pattern of release. This effect was not observed in diestrus rats. The ovariectomy abolished the facilitatory effect of allopregnanolone on NMDA-evoked 2 [³H]dopamine release.5. Subcutaneous administration of exogenous estrogen (25 mg/rat) and progesterone (1 mg/rat) restored the facilitatory effect on dopaminergic input.6. These results suggest that allopregnanolone is a neurosteroid able to modulate dopamine release in an ovarian-hormone-fluctuation-dependent manner and provide further support for a role of allopregnanolone as a modulator of glutamatergic–dopaminergic interaction in the corpus striatum.     

Food choice of Antarctic soil arthropods clarified by stable isotope signatures

Antarctic soil ecosystems are amongst the most simplified on Earth and include only few soil arthropod species, generally believed to be opportunistic omnivorous feeders. Using stable isotopic analyses, we investigated the food choice of two common and widely distributed Antarctic soil arthropod species using natural abundances of ¹³C and ¹⁵N and an isotope labelling study. In the laboratory we fed the isotomid springtail Cryptopygus antarcticus six potential food sources (one algal species, two lichens and three mosses). Our results showed a clear preference for algae and lichens rather than mosses. These results were corroborated by field data comparing stable isotope signatures from the most dominant cryptogams and soil arthropods (C. antarcticus and the oribatid mite Alaskozetes antarcticus). Thus, for the first time in an Antarctic study, we present clear evidence that these soil arthropods show selectivity in their choice of food and have a preference for algae and lichens above mosses.     

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of human and murine isoforms of UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine 2-epimerase/<Emphasis Type="Italic">N</Emphasis>-acetylmannosamine kinase (GNE)

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.     

Effect of competition on stable carbon isotope ratios of two tussock grass species

Summary Previous studies have shown that plant carbon isotope composition varies when plants experience differences in water and nutrient availability. However, none have addressed the effect of root interactions, including competition for these soil resources, on carbon isotope ratios. We studied the effect of interspecific root interactions on the productivity and carbon isotope ratios of two Great Basin tussock grass species (Agropyron desertorum and Pseudoroegneria spicata). We compared grasses grown in mixture with sagebrush (Artemisia tridentara) to grasses in similar mixtures but where root interactions with sagebrush were limited by fiberglass partitions. During both years of the study, tussocks growing in competition with sagebrush produced tissue with more negative ¹³C values than grasses experiencing limited root interaction with sagebrush. The magnitude of this difference (0.5 to 0.9%) is similar to that found in other studies when soil fertility and moisture availability were altered.     

N-[2-Naphthyl]-glycine hydrazide,a potent inhibitor of DNA-dependent RNA polymerase ofMycobacterium tuberculosis H37RV

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) ofMycobacterium tuberculosis H₃₇R_v. At a concentration of 10^-9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the λ_max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus,Microsporum canis. At a concentration of 10⁻⁹ M it inhibits RNA polymerase II (32%) but not RNA polymerasesI andIII     

Solution-mediated syntheses and characterizations of two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3]

Two new zincophosphites [C₆H₁₄N₂]_0.5[Zn(H₂PO₃)₂] 1 and [C₄H₁₂N₂]_0.5[(CH₃)₂NH₂][Zn₂(HPO₃)₃] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO₄ and HPO₂(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO₄ and HPO₃ units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.     

Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac₂ and theN-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound theN-formyl derivative, whereas aN-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.Dedicated to Professor Dr Gerhard Uhlenbruck on the occasion of his 65th birthday.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.