Denitration of carbohydrate α-nitroepoxides by nucleophiles

Several nucleophiles were separately treated with methyl and phenyl 2,3-anhydro-4,6-O-benzylidene-3-deoxy-3-nitro-β-d-allopyranoside, to give 2-substituted aldos-3-ulose derivatives. In the latter case, the subsequent β-elimination of the aglyconic phenyl group always occurred to afford the corresponding glycal. Reaction mechanisms thereof are also discussed.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant α-1-antitrypsin

α-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50–60% as much α-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. When analyzed in the ultracentrifuge, the S-type α-1-antitrypsin exhibited a molecular weight of 47500 which was essentially the same as that of the M-type (47300) and the Z-type (47500) α-1-antitrypsin. The S-type α-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type α-1-antitrypsin. In addition, M- and S-type α-1-antitrypsin have very similar amino acid compositions.     

Reactions of Re(III) with α-diketones: Oxidative addition

The reactions of [ReCl₃(CH₃CN)(PPh₃)₂] with benzil PhC(O)C(O)Ph, and with a natural 1,2-naphthoquinone derivative, β-lapachone (Lap), result in oxidative addition with the formation of Re(V) complexes with stilbenediolate, [ReCl₃(PhC(O)C(O)Ph)(PPh₃)] (1) and with a reduced semiquinonic form of lapachone, [Re^IVCl₃(Lap⁻)(PPh₃)] (2). The structures of both compounds were established by X-ray crystallography.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Maternal effects and carbohydrate changes of Pinus pinaster after inoculation with Fusarium circinatum

Key message

Carbohydrate differences in offspring as a consequence of maternal effects explain transgenerational tree-pathogen interactions.

Abstract

The expression of disease is increasingly recognised as being influenced by maternal effects, given that environmental conditions experienced by mother trees affect tolerance in offspring. It is hypothesised that plant carbohydrates could mediate transgenerational tree-pathogen interactions. The carbohydrate content of Pinus pinaster seedlings obtained from two contrasting maternal environments was studied and seedlings from the two environments were challenged with Fusarium circinatum. The representative mid-infrared spectra of samples in the range of the carbohydrates diagnosed higher proportion of methylesterified pectic polysaccharides and lower proportion of nonesterified pectic polysaccharides for inoculated than for control seedlings. Total carbohydrate content of seedlings from the unfavourable environment did not differ much from total carbohydrate content of seedlings from the favourable maternal environment. However, glucose was 13 % higher and uronic acids 11 % lower in seedlings from the favourable environment after inoculation in comparison to seedlings from the unfavourable maternal environment which had their carbohydrate contents unaltered after inoculation. It is concluded that plant carbohydrates mediate transgenerational tree-pathogen interactions.     

Reaction of Toxic Bicyclic Phosphates with Acetylcholinesterases and α-Chymotrypsin

Some toxic bicyclic phosphates (BPs) inhibited acetylcholinesterases (AChEs), but the activity was very weak. Even the most potent inhibitor, 4-nitro BP, inhibited bovine erythrocyte and housefly head AChEs by only 37 and 38 per cent, respectively, at 1.5 mm. Kinetic analysis indicated that the poor inhibitory activity of 4-nitro BP is ascribed not only to the low affinity for AChEs but also to its poor phosphorylating ability. Similar findings were obtained in the case of the reaction of BPs with the serine enzyme α-chymotrypsin. Despite the relatively high reactivity in an alkaline solution, BPs are much less active than other bioactive organophosphorus esters in phosphorylating a general-base-catalyzed hydroxyl group. This fact suggests that the toxic action of BPs does not result from the phosphorylation of a critical site in biological systems.     

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.     

The carbohydrate–polypeptide linkages, the amino acid sequences of the peptides adjacent to some of these bonds, and the composition and size of the carbohydrate units of α1-acid glycoprotein

1. The glycopeptides derived from a proteolytic digest of sialic acid-free α₁-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that α₁-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH₂)-Pro-Lys; Thr-Asp(NH₂)-Ala; Asp(NH₂)-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the -amino group of lysine are free and that the β-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate–polypeptide linkages of at least four carbohydrate units of α₁-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of α₁-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 (Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl_z or cytochrome b-559.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

Molecular Modeling and Physicochemical Properties of Supramolecular Complexes of Limonene with α- and β-Cyclodextrins

This study evaluated three different methods for the formation of an inclusion complex between alpha- and beta-cyclodextrin (α- and β-CD) and limonene (LIM) with the goal of improving the physicochemical properties of limonene. The study samples were prepared through physical mixing (PM), paste complexation (PC), and slurry complexation (SC) methods in the molar ratio of 1:1 (cyclodextrin:limonene). The complexes prepared were evaluated with thermogravimetry/derivate thermogravimetry, infrared spectroscopy, X-ray diffraction, complexation efficiency through gas chromatography/mass spectrometry analyses, molecular modeling, and nuclear magnetic resonance. The results showed that the physical mixing procedure did not produce complexation, but the paste and slurry methods produced inclusion complexes, which demonstrated interactions outside of the cavity of the CDs. However, the paste obtained with β-cyclodextrin did not demonstrate complexation in the gas chromatographic technique because, after extraction, most of the limonene was either surface-adsorbed by β-cyclodextrin or volatilized during the procedure. We conclude that paste complexation and slurry complexation are effective and economic methods to improve the physicochemical character of limonene and could have important applications in pharmacological activities in terms of an increase in solubility.     

Reactions of nonsymmetrical bidentate linkers having a phthalimidoyl and acid chloride group or a 2-benzothiazole group with α-D-glucoside derivatives and their application for the modification of cellulose

The reaction of ‘active ester’ bidentate cross-linking reagents, phthalimido 4-chloroformylbutanoate (1) and phthalimido 4-(2-benzothiazolyloxycarbonyl)butanoate (2) with several protected d-glucose derivatives is described. These reactions are used to introduce the reactive group into cellulose (filter paper) with the aim of linking proteins and cellulose.     

Highly stereoselective C-α-d-ribofuranosylation. Reactions of d-ribofuranosyl fluoride derivatives with enol trimethylsilyl ethers and with allyltrimethylsilane

2,3,5-Tri-O-methyl-d-ribofuranosyl flouride (6), 2,3-di-O-benzyl-5-O-methyl-d-ribofuranosyl fluoride (7), and 5-O-benzyl-2,3-di-O-methyl-d-ribofuranosyl fluoride (8) were obtained in 57 (6α, 15; and 6β, 42), 87 (7α, 22; and 7β, 65), and 85.5 (8α, 35.5; and 8β, 50%) yields, respectively, from the corresponding OH-1 derivatives by the reaction with N,N-diethyl-1,1,2,3,3,3-hexafluoropropylamine, adduct of hexafluoropropene with diethylamine. These fluorides and 2,3,5-tri-O-benzyl-d-ribofuranosyl fluoride (5) reacted with isopropenyl trimethylsilyl ether, (Z)-1-ethyl-1-propenyl trimethylsilyl ether, and allyltrimethylsilane, in the presence of boron trifluoride·diethyl etherate to give the corresponding 1-d-ribofuranosyl-2-propanones, 2-d-ribofuranosyl-3-pentanones, and 3-d-ribofuranosyl-1-propenes in good yields. C-Acetonylation was confirmed to afford the α-d anomer as the initial product, and the α-d anomer was isomerized into the corresponding β-d anomer to give a mixture. The C-allylation reaction gave only the α-d anomer. C-Pentanonylation, however, gave a mixture of diastereoisomers that could not be isolated. All reactions afforded almost the same results starting with either α- or β-d-ribofuranosyl fluoride. No reaction of the β anomer of 5 with 1-isopropyl-2-methyl-1-propenyl trimethylsilyl ether took place.     

Enhancement of mycobactericidal activity of glutaraldehyde with α,β-unsaturated and aromatic aldehydes

Summary Several ,-unsaturated and aromatic aldehydes were evaluated for antimicrobial activity usingMycobacterium bovis as the test strain. Activity of most of the compounds was determined in the presence and absence of 2% glutaraldehyde. Several compounds highly active against this organism, e.g. 2-pentenal, benzaldehyde, ando-phthalaldehyde showed rapid kill of >10⁵ CFU ml^–1 in 5 min. Activity of ,-unsaturated compounds substituted in the ₁ position showed increasing activity with increasing chain length. Of the aromatic aldehydes tested, benzaldehyde andp-dimethylamino benzaldehyde showed little activity alone, but when combined with 2% glutaraldehyde showed increased activity. Substituents added to the benzaldehyde ring (nitro, chloro, methyl, and methoxy) all detracted from the synergism, but still showed increased activity over the activity of 2% glutaraldehyde. The same affect was noted with disubstituted benzaldehyde compounds but not with substitutedo-phthaladehyde (2-formylformaldehyde).     

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to α_V integrins. The interaction of HPEV1 with α_V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins α_Vβ₃ and α_Vβ₆ to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, α_Vβ₆ bound more efficiently than α_Vβ₃ to immobilized HPEV1. Moreover, soluble α_Vβ₆, but not α_Vβ₃, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4, 19, 24, 30, 38, 39, 51, 58, 78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (N-terminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5′ untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20, 28, 42, 73, 80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infections (1, 8, 10, 28, 80). Currently, the role of the unique molecular, structural, and antigenic characteristics of HPEVs in the pathogenesis of infection is unknown.HPEV types 1, 2, 4, 5, and 6 are known to possess an RGD motif near the C terminus of VP1 that is known to facilitate binding of cellular ligands (e.g., fibronectin) to α_v integrins. The motif is in an analogous position to motifs in coxsackievirus A9 (CAV9) and echovirus 9 (EV9; Barty strain) (Fig. ​(Fig.1).1). The role of the RGD sequence in cellular entry and subsequent replication of HPEV1 has been shown through blocking assays with RGD-containing peptides, mutation of the sequence, and function-blocking antibodies to α_v integrins (11, 43, 62, 71). These results strongly suggested that α_v integrins play a central role in the initiation of HPEV1 infection. Direct involvement of α_v integrins in the infectious entry of HPEV1 was further confirmed by overexpression of human α_vβ₁ and α_vβ₃ integrins in Chinese hamster ovary (CHO) cells, allowing successful virus infection (74). There are no reports yet on the identification of receptors for the HPEV types lacking the RGD motif (HPEV3, HPEV7, and HPEV8) (19, 39, 51).Open in a separate windowFIG. 1.Sequence alignments. Amino acid sequence alignment of the viral coat protein VP1 from different picornaviruses with the CAV9 secondary structure derived from the atomic model displayed above the alignment (34). The columns boxed in blue with red letters signify similarity, and the red column signifies identity. There is limited similarity between HPEV and other picornaviruses. C-terminal RGD motifs are boxed in red.Although the crystal structures of several picornaviruses have been determined (3, 26, 34, 35, 44, 57, 59, 65, 68, 72) and the receptor interactions have been studied in detail by X-ray crystallography, electron cryo-microscopy (cryo-EM), and three-dimensional (3D) image reconstruction (6, 9, 23, 31, 32, 47, 83), there is no structural information available for the parechoviruses or parechovirus-receptor complexes. Here, we compare the binding of α_Vβ₃ and α_Vβ₆ to HPEV1 in vitro by biochemical assays and determine the structures of HPEV1 and the corresponding HPEV1-integrin complexes.     

Far-infrared Bands of Amino-acid Residues with α-Helical and β-Form Conformations

INFRARED spectra in the far-infrared region are very sensitive to the conformational changes of polypeptide backbones^1–6. We have measured the infrared spectra of sequential polypeptides in the region from 700 to 200 cm^?1 and have found several far-infrared bands characteristic of component amino-acid residues with α-helical and β-form conformations.