Efficient synthesis of 6-<Emphasis Type="Italic">O</Emphasis>-palmitoyl-1,2-<Emphasis Type="Italic">O</Emphasis>-isopropylidene-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucofuranose in an organic solvent system by lipase-catalyzed esterification

In order to synthesize a sugar ester at high concentration, 1,2-O-isopropylidene-α-d-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value (240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1,200 mmol palmitic acid/kg solvent were used in this solvent mixture.     

A computational investigation on the antioxidant potential of myricetin 3,4′-di-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">α</Emphasis>-<Emphasis Type="Italic">L</Emphasis>-rhamnopyranoside

In this work, we present a computational study on the antioxidant potential of myricetin 3,4\(^{\prime }\)-di-O-α-L-rhamnopyranoside (Compound M). A density functional theory (DFT) approach with the B3LYP and LC-ωPBE functionals and with both the 6-311G(d,p) and 6-311+G(d,p) basis sets was used. The focus of the investigation was on the structural and energetic parameters including both bond dissociation enthalpies (BDEs) and ionization potentials (IPs), which provide information on the potential antioxidant activity. The properties computed were compared with BDEs and IPs available in the literature for myricetin, a compound well known for presenting antioxidant activity (and the parent molecule of the compound of interest in the present work). Myricetin 3,4\(^{\prime }\)-di-O-α-L-rhamnopyranoside presented the lowest BDE to be 79.13 kcal/mol (as determined using B3LYP/6-311G(d,p) in water) while myricetin has a quite similar value (within 3.4 kcal/mol). IPs computed in the gas phase [B3LYP/6-311G(d,p)] are 157.18 and 161.4 kcal/mol for myricetin 3,4\(^{\prime }\)-di-O-α-L-rhamnopyranoside and myricetin, respectively. As the values of BDEs are considerably lower than the ones probed for IPs (in the gas phase or in any given solvent environment), the hydrogen atom transfer mechanism is preferred over the single electron transfer mechanism. The BDEs obtained suggest that myricetin 3,4\(^{\prime }\)-di-O-α-L-rhamnopyranoside can present antioxidant potential as good as the parent molecule myricetin (a well-known antioxidant). Therefore, experimental tests on the antioxidant activity of Compound M are encouraged.     

Enzymatic synthesis of alkyl glucosides using <Emphasis Type="Italic">Leuconostoc</Emphasis> <Emphasis Type="Italic">mesenteroides</Emphasis> dextransucrase

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants. Young-Min Kim and Byung-Hoon Kim contributed equally to this work.     

Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors of <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine 4-sulfate 6-<Emphasis Type="Italic">O</Emphasis>-sulfotransferase

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC₅₀ value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">Z</Emphasis>, <Emphasis Type="Italic">N</Emphasis>′-Formyl α-Amino Acid Derived Gem-Diamines

A variety of N-carbobenzoxy, N′-formyl gem-diaminoalkyl derivatives have been obtained through Goldsmith-Wick reaction of Z-α-amino acid/peptide acid derived isocyanates with 96% HCOOH in presence of 4-dimethylaminopyridine (DMAP) as catalyst. The reaction proceeds to completion within 2–4 h and results in good yields of the products isolated as stable solids.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

Oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.     

Syntheses of mucin-type <Emphasis Type="Italic">O</Emphasis>-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of <Emphasis Type="Italic">Bifidobacterium</Emphasis> endo-α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Identification of aryl-phospho-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidases in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

Four aryl-phospho--d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho--d-glucopyranoside as a substrate. Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho--d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes. Together, these four genes account for >99.9% of the glucosidase activity in B. subtilis on aryl-phospho--d-glucosides. yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl--d-glucosides. ydhP was also not induced by aryl--d-glucosides. However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth. Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl--d-glucosides. However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl--d-glucosides as the sole carbon source.Abbreviations MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--d-glucopyranoside - MUGal 4-Methylumbelliferyl--d-galactopyranoside - MUG-P 4-Methylumbelliferyl--d-glucopyranoside-6-phosphate     

The Effect of Exogenous β-<Emphasis Type="Italic">N-</Emphasis>Methylamino-<Emphasis Type="SmallCaps">l</Emphasis>-alanine on the Growth of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

β-N-Methylamino-l-alanine (BMAA), a non-proteinogenic amino acid, has been detected in a range of cyanobacteria, including terrestrial, aquatic, free living and endosymbiotic species. The widespread occurrence of cyanobacteria in the environment raises concerns regarding the ecological and toxicological impact of BMAA, and consequently, studies have focussed extensively on the toxicity and environmental impact of BMAA, while no research has addressed the ecophysiological or metabolic role of the compound in cyanobacteria. In this study, both the uptake of exogenous BMAA by and the effect of exogenous BMAA on the growth of Synechocystis PCC6803 were investigated. BMAA was rapidly taken up by the non-diazotrophic cyanobacterium Synechocystis PCC6803 in a concentration dependent manner. The presence of exogenous BMAA resulted in a substantial and concentration-dependent decrease in cell growth and the substantial loss of photosynthetic pigmentation. Similar effects were seen in the presence of the non-proteinogenic amino acid, 2,4-diaminobutyric acid but to a lesser degree than that of BMAA. The effects were reversed when light was decreased from 16 to 10 μmol m⁻² s⁻¹. Control cultures grown in the presence of l-arginine, l-asparagine, l-glutamate and glycine showed normal or slightly increased growth with no change in pigmentation. The decrease in growth rate coupled to bleaching indicates that BMAA may induce chlorosis in the presence of adequate photosynthetic radiation suggesting a connection between BMAA and the induction of conditions, such as nitrogen or sulphur depletion, that result in growth arrest and the induction of chlorosis.     

New furano- and pyrrolo[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidine nucleosides and their 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphates: Synthesis and biological properties

Bicyclic furano[2,3-d]pyrimidine ribonucleosides were synthesized by Pd(0)-and CuI-catalyzed coupling of 5-iodouridine with terminal alkynes. The treatment of the resulting nucleosides with ammonia or methylamine solution in aqueous alcohol resulted in pyrrolo-and N ⁷-methylpyrrolo[2,3-d]pyrimidine nucleosides. 5′-O-Triphosphates of bicyclic nucleosides were obtained by the treatment of the nucleosides with POCl₃ in the presence of a “proton sponge.” The 5′-O-triphosphates are not substrates for HCV RNA-dependent RNA polymerase, but are effective substrates for HCV RNA helicase/NTPase and did not inhibit ATP hydrolysis. Only 3-(β-D-ribofuranosyl)-6-decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one showed a moderate anti-HCV activity in the HCV replicon system and efficiently inhibited replication of bovine viral diarrhea virus (BVDV) in KCT-cells, other compounds being inactive. None of the compounds were cytotoxic within the tested range of concentrations.     

Di-<Emphasis Type="Italic">tert</Emphasis>-butylsilylene-directed α-selective synthesis of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl T-antigen analogues

Seven analogues of p-nitrophenyl T-antigen [Galβ(1→3)GalNAcα(1→O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-α-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcβ(1→3){GlcNAcβ(1→6)}GalNAcα(1→O)PNP [core 4 type], GalNAcα(1→3)GalNAcα(1→O)PNP [core 5 type], GlcNAcβ(1→6)GalNAcα(1→O)PNP [core 6 type], GalNAcα(1→6)GalNAcα(1→O)PNP [core 7 type], Galα(1→3)GalNAcα(1→O)PNP [core 8 type], Glcβ(1→3)GalNAcα(1→O)PNP and GalNAcβ(1→3)GalNAcα(1→O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed α-galactosylation as a key reaction.     

Association of polymorphism of the β(1, 4)-<Emphasis Type="Italic">galactosyltransferase</Emphasis>-<Emphasis Type="Italic">I</Emphasis> gene with milk production traits in Holsteins

The β(1,4)-galactosyltransferase-I gene (β4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the β4galt1 genotype. We concluded that the β4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Indel patterns of the plastid DNA<Emphasis Type="Italic"> trnL</Emphasis>–<Emphasis Type="Italic">trnF</Emphasis> region within the genus<Emphasis Type="Italic"> Poa</Emphasis> (Poaceae)

The use of insertion/deletion (indel) patterns from sequences of the trnL intron and trnL–F intergenic spacer (IGS) in finding plastid genome types of the genus Poa L. was studied. New sequences for 23 taxa (P. alpina, P. badensis, P. bulbosa, P. crassipes, P. molinerii, P. annua, P. chaixii, P. granitica, P. pratensis, P. sibirica, P. remota, P. botryoides, P. cenisia, P. compressa, P. laxa, P. margilicola, P. media, P. nemoralis, P. palustris, P. pannonica, P. pirinica, P. riphaea, and P. sejuncta) and 18 previously published sequences, which represent 11 of the 13 sections listed for Poa in Flora Europaea, were investigated. Collections were made primarily in central Europe. Indel patterns, despite sampling less than 0.7% of the plastid genome, produced four taxa groupings that were congruent with the major divisions obtained in intensive, previously published restriction-site studies. Insertion/deletion events in the trnL intron and trnL–trnF IGS were in nearly all cases unique to a single pattern group and thus provided almost no information about relationships among these groups. Indels did, however, provide a meaningful infrageneric classification criterion for Poa. They can serve as useful tools in studying relationships within this genus.