Induction of T → G and T → A transversions by 5-formyluracil in mammalian cells

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5′-CFTAAG-3′ and 5′-CTFAAG-3′ (F represents 5-fU), the recognition site for the restriction enzyme AflII (5′-CTTAAG-3′). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01–0.04%. The T → G and T → A transversions were the mutations found most frequently, suggesting the formation of 5-fU·C and 5-fU·T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.     

Probe design and synthesis of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of N-glycan

Synthesis and clusterization of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of the N-glycan, as the molecular probes for their biological evaluation, are reported. Key step is the quantitative and the completely α-selective sialylation of the C5-azide N-phenyltrifluoroacetimidate with the disaccharide acceptor, Galβ(1→3)GlcNTroc. Clusterization of the 16 molecules of trisaccharide motif was also achieved by the ‘self-activating click reaction’. These probes could efficiently be labeled by biotin and/or other fluorescence- or radioactive reporter groups through either cross metathesis, acylation, Cu(I)-mediated Huisgen [2+3]-cycloaddition, or the azaelectrocyclization to utilize the various biological techniques.     

Crystal and molecular structure of methyl L-glycero-α-D-manno-heptopyranoside, and synthesis of 1→7 linked L-glycero-D-manno-heptobiose and its methyl α-glycoside

Methyl l-glycero-α-d-manno-heptopyranoside was synthesized in good yield by a Fischer-type glycosylation of the heptopyranose with methanol in the presence of cation-exchange resin under reflux and microwave conditions, respectively. The compound crystallized from 2-propanol in an orthorhombic lattice of space group P2(1)2(1)2 showing a comparatively porous structure with a 2-dimensional O-H?O hydrogen bond network. As model compounds for the side chain domains of the inner core structure of bacterial lipopolysaccharide, l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-d-manno-heptopyranose and the corresponding disaccharide methyl α-glycoside were prepared. The former compound was generated via glycosylation of a benzyl 5,6-dideoxy-hept-5-enofuranoside intermediate followed by catalytic osmylation and deprotection. The latter disaccharide was efficiently synthesized in good yield by a straightforward coupling of an acetylated N-phenyltrifluoroacetimidate heptopyranosyl donor to a methyl 2,3,4,6-tetra-O-acetyl heptopyranoside acceptor derivative followed by Zemplén deacetylation.     

Synthesis of an Isoglutathione Derivative by Proteases and Its α→γ Transpeptidation

Benzyloxycarbonyl-l-cysteine and glycine benzhydrylamide were condensed by papain in a yield of 39.5%. After elimination of the N-protecting group, l-cysteinylglycine benzhydrylamide was condensed with benzyloxycarbonyl-l-glutamic acid by acid protease from Irpex lacteus Fr. in a yield of 21.0%. From the isoglutathione derivative thus obtained, a γ-linkage between glutamic acid and cysteine was formed by α → γ transpeptidation in alkaline conditions after esterification of γ-carboxylic acid.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

NMR and conformational studies of linear and cyclic oligo-(1→6)-β-D-glucosamines

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β-D-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants (3)J(C,H) and (3)J(H,H). Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5-C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.     

Effects of β1 →4 Linked Galactooligosaccharides on Use of Magnesium and Calcification of the Kidney and Heart in Rats Fed Excess Dietary Phosphorus and Calcium

Magnesium deficiency was induced in male Wistar rats by adding an excess of phosphorus and calcium to the diet (1.195 g of phosphorus and 1.04g of calcium/100g of diet). Feeding of these animals with a diet containing β1→4 linked galactooligosaccharides (4′-GOS) (5g of 4′-GOS/100g of diet) increased the apparent magnesium absorption ratios and the concentrations of magnesium in the serum and femur, and reduced accumulation of calcium in the kidney and heart. We speculate that the use of magnesium increased by feeding 4′-GOS to a limited extent prevented the lower magnesium status and the severity of calcification of the kidney and heart caused by excess dietary phosphorus and calcium.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Molecular and crystal structure of the regenerated form of (1→3)-α-d-glucan

The crystal structure of a regenerated form of (1→3)-α-d-glucan, obtained by solid state deacetylation of the triacetate derivative, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. The structure crystallizes in an orthorhombic unit cell with parameters $\text{a = 16.46}\text{A}\text{?}\text{, b = 9.55}\text{A}\text{?}$ and c (fibre repeat)=8.44 Å, and space group P2₁2₁2₁. The chain conformation is nearly completely extended and is very close to a 2/1 helix, even though the dimer residue is the crystallographic repeat unit. An intramolecular O(2)  O(4)′ hydrogen bond stabilizes the conformation and extensive intermolecular hydrogen-bonding abilizes the packing. The resulting structure is sheet-like, with an alternating polarity of chain directions within the sheet. In its sheet-like character, extensive hydrogen-bonding, and insolubility in water, this polymorph of (1→3)-α-d-glucan resembles regenerated cellulose. The reliability of the structure analysis is indicated by the X-ray residual R=0.206.     

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

Intracellular and cell wall associated (1 → 3) β glucanases of Saprolegnia

Summary Fractionation of proteins of mycelial cell free extracts from Saprolegnia monoica revealed the presence of two different (1.3) glucanases. The most important fraction exhibited activity against laminarin and p. nitrophenyl BD. glucopyranoside. The other was active on both laminarin and oxidized laminarin. This endo-glucanase represented the main part of glucanase activities released during cell wall autolysis. Properties and cellular distribution of these enzymes are discussed in respect to their morphogenetic role in hyphal differentiation.     

A comparative analysis of the spin state distribution of in vitro and in vivo mutants of PsaC. A biochemical argument for the sequence of electron transfer in Photosystem I as FX → FA → FB → ferredoxin/flavodoxin

The EPR properties of in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants of PsaC are compared in an attempt to extract information about electron transfer not contained in any one of these studies in isolation. This analysis indicates that 1) sulfur from an external 'rescue thiolate' is preferred over oxygen from an aspartate or serine replacement amino acid as a ligand to the F_A and F_B iron-sulfur clusters; 2) the inherent spectroscopic symmetry in the F_A and F_B clusters of unbound PsaC is lost when PsaC is docked to its site on the PsaA/PsaB heterodimer; 3) the bound 'rescue thiolate' ligand in the modified site of the F_A cluster, but not the F_B, cluster is displaced when PsaC is docked to its site on the PsaA/PsaB heterodimer; 4) the free energy of binding PsaC to the PsaA/PsaB heterodimer drives the otherwise-unfavorable ligand replacement in the F_A site. These and other findings argue that the substitute ligands support a [4Fe–4S] cluster at the modified site, but the cluster is in either a ground spin state of S 3/2 or S = 1/2 depending on the chemical identity of the ligand, on whether PsaC is unbound or bound, and on the reduction state of the cluster in the unmodified site. By a comparative analysis of the spin state distribution of the in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants, and with knowledge from the X-ray crystal structure that PsaC is bound asymmetrically to the PS I reaction center, an independent case is made that PsaC is oriented so that the F_A cluster is proximal to F_X and the F_B cluster is distal to F_X. These results are compared and contrasted with the results of in vivo mutagenesis studies of PsaC in Anabaena variabilis ATCC 29413 and in Chlamydomonas reinhardtii. In all cases, the primary data can be interpreted to support the sequence of electron transfer as F_X F_A F_B ferredoxin.     

Differential binding characteristics and applications of dGalβ1→3dGalNAc specific lectins

Summary The binding properties of Arachis hypogaea (PNA), Bauhinia vurpurea alba (BPL), Maclura pomifera (MPL) and Sophora japonica (SJL) lectins were studied by quantitative precipitin and precipitin inhibition assays, demonstrating them to be most specific for dGal13dGalNAc residues. Additionally, each lectin had its own binding characteristic such as different binding abilities to dGal14dGlcNAc or dGal13dGlcNAc1linked oligosaccharides, and/or dGalNAc1linked to the Ser or Thr of the protein moiety. These differential binding characteristics can be used for investigating fine differences of the carbohydrate structure of the glycoconjugates, especially those having dGal13dGalNAc residues as terminal non-reducing ends.Abbreviations dGal d-galactopyranose - dMan d-mannopyranose - dGalNAc 2-acetamido-2-deoxy-d-galacto-pyranose - dGlcNAc 2-acetamino-2-deoxy-d-glucopyranose - LFuc L-fucose - NeuNAc N-acetylneuraminic acid - Ser serine - Thr Threonine - RCA Ricinus communis agglutinin - SBA Soy bean agglutinin (Glycine max) - HPA Helix pomatia agglutinin - DBL Dolichos biflorus lectin - GCL Geodia cydonium lectin     

Structural and electronic properties of Z isomers of (4α→6´´,2α→O→1´´)-phenylflavans substituted with R = H, OH and OCH₃ calculated in aqueous solution with PCM solvation model

In the search for new antioxidants, flavan structures called our attention, as substructures of many important natural compounds, including catechins (flavan-3-ols), simple and dimeric proanthocyanidins, and condensed tannins. In this work the conformational space of the Z-isomers of (4α→6′′, 2α→O→1′′)-phenylflavans substituted with R = H, OH and OCH₃ was scanned in aqueous solution, simulating the solvent by the polarizable continuum model (PCM). Geometry optimizations were performed at B3LYP/6-31 G** level. Electronic distributions were analyzed at a better calculation level, thus improving the basis set (6-311++G**). A topological study based on Bader′s theory (atoms in molecules) and natural bond orbital (NBO) framework was performed. Furthermore, molecular electrostatic potential maps (MEPs) were obtained and thoroughly analyzed. The stereochemistry was discussed, and the effect of the solvent was addressed. Moreover, intrinsic properties were identified, focusing on factors that may be related to their antioxidant properties. Hyperconjugative and inductive effects were described. The coordinated NBO/AIM analysis allowed us to rationalize the changes of MEPs in a polar solvent. To investigate the molecular and structural properties of these compounds in biological media, the polarizabilities and dipolar moments were predicted which were further used to enlighten stability and reactivity properties. All conformers were taken into account. Relevant stereoelectronic aspects were described for understanding the stabilization and antioxidant function of these structures.     

Effects of pH,light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.     

Interaction of (1→4)- and (1→6)-linked disaccharides with the fenton reagent under physiological conditions

Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.     

Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.     

DFTr studies of five- and six-residue cyclic-β(1→4) cellulosic molecules

Density functional (DFT) conformational in vacuo studies of cellobiose have shown that ?_H‐anti conformations are low in energy relative to the syn forms, while the ψ_H‐anti forms are higher in energy. Further, as the cellulosic fragments became larger than a disaccharide and new hydrogen bonding interactions between multiple residues become available, stable low energy ?_H‐anti, and ψ_H‐anti cellulosic structures became possible. To test the stability of cyclic anti‐conformations, a number of β‐linked five‐ and six‐residue molecules were created and then energy optimized in solvent (water, n‐heptane) using the implicit solvation method COSMO at the B3LYP level of theory. The created symmetric cyclic structures were without distortion. Upon optimization some cyclic conformations were found to be of low energy when compared with linear five‐ and six‐residue chains, after correcting the energy for the exclusion of a water molecule upon cyclization. It was also obvious from the hydrogen bonding network formed above and below the plane of the cyclic structure that these structures could exhibit strong synergistic tendencies. The conformational energy preferences for clockwise “c” and counter‐clockwise “r” hydroxyl groups and preference for the hydroxymethyl rotamers is described. Because these structures contain energetically unfavorable flipped conformations in water, that is, dihedral angles of ～180°/0° or ～0°/180° in ?_H/ψ_H, it is clear that the synthesis of these compounds will be challenging. © 2012 Wiley Periodicals, Inc. Biopolymers 97:568–576, 2012.