Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.     

Characterization of new glycolipid biosurfactants,tri-acylated mannosylerythritol lipids,produced by <Emphasis Type="Italic">Pseudozyma</Emphasis> yeasts

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4′,6′-di-O-acetyl-2′,3′-di-O-alka(e)noyl)-β-d-mannopyranosyl]-d-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Enhanced benzophenanthridine alkaloid production and protein expression with combined elicitor in <Emphasis Type="Italic">Eschscholtzia californica</Emphasis> suspension cultures

Production of the benzophenanthridine alkaloids in Eschscholtzia californica suspension cell cultures was optimized by adding 0.5 mg methyl jasmonate (MJ) and 0.02 mg salicylic acid (SA)/g FCW after 7 days cultivation. Sanguinarine reached 24 mg/g DCW by such treatment; 10 times higher than in control cell cultures. MJ and SA induced expression of berberine bridge enzyme and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase, respectively. MJ plus SA induced over-expression of both enzymes.     

Isolation of a new flavonol glycoside and its effects on the blue color of seed coats ofOphiopogon jaburan

From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10⁻³ M OK-2 and 2.5×10⁻³ M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.     

A new acylated flavonol diglycoside from <Emphasis Type="Italic">Atriplex littoralis</Emphasis>

A new acetylated flavonol glycoside: patuletin 3-O-[5′″-O-feruloyl-β-D-apiofuransyl (1′″→2′′)-β-D-glucopyranoside] (2), together with a known patuletin 3-O-β-D-glucopyranoside (1) were isolated from the aerial part of Artiplex littoralis L. (Chenopodiacease). Their structures were elcidated by acid hydrolysis and spectroscopic methods including UV, ¹H, ¹³C NMR and ESI-MS for both compounds, additionally 2D-NMR, HSQC, HMBC experiments were performed for 2.     

Efficient production of mannosylerythritol lipids with high hydrophilicity by <Emphasis Type="Italic">Pseudozyma hubeiensis</Emphasis> KM-59

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-d-erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l⁻¹ day⁻¹. We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella () in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.     

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-M<Subscript>r</Subscript> apoplastic inhibitor

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H₂O₂ in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-α-L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-D-xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl−¹⁴C]FAXX and [pentosyl−³H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [¹⁴C]feruloyl groups were converted to [¹⁴C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H₂O₂-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H₂O₂ and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M_r, was destroyed by heating, and remained in the aqueous phase (pH ≈3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.     

Accumulation of cyclohexenone derivatives in barley, wheat and maize roots in response to inoculation with different arbuscular mycorrhizal fungi

Glomus intraradices , Glomus mosseae, and Gigaspora rosea leads to the accumulation of cyclohexenone derivatives. Mycorrhizal roots of all plants accumulate in response to all three fungi blumenin [9-O-(2′-O-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one], 13-carboxyblumenol C 9-O-gentiobioside, nicoblumin [9-O-(6′-O-β-glucopyranosyl)-β-glucopyranoside of 13-hydroxy-6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] and another, as yet unidentified, cyclohexenone derivative. The accumulation of all four compounds in three tested mycorrhizal plants colonized by the three arbuscular mycorrhizal fungi indicates no fungus-specific induction of these compounds. Accepted: 6 October 1999     

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcusalbus

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-d-galactopyranosyl-d-mannose (epilactose). Based on the sequence alignment with N-acetyl-d-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)₆ core barrel structure.     

Structural characterization and surface-active properties of a new glycolipid biosurfactant,mono-acylated mannosylerythritol lipid,produced from glucose by <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis>

Mannosylerythritol lipids (MELs), which are glycolipid biosurfactants produced by Pseudozyma yeasts, show not only excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from glucose as the sole carbon source, P. antarctica was found to produce unknown glycolipids more hydrophilic than conventional “di-acylated MELs,” which have two fatty acyl esters on the mannose moiety. Based on a detailed characterization, the most hydrophilic one was identified as 4-O-(3′-O-alka(e)noyl-β-d-mannopyranosyl)-d-erythritol namely, “mono-acylated MEL.” The mono-acylated MEL reduced the surface tension of water to 33.8 mN/m at a critical micelle concentration (CMC) of 3.6 × 10⁻⁴ M, and its hydrophilic–lipophilic balance was tentatively calculated to be 12.15. The observed CMC was 100-fold higher than that of the MELs hitherto reported. Interestingly, of the yeast strains of the genus Pseudozyma, only P. antarctica and P. parantarctica gave the mono-acylated MEL from glucose, despite a great diversity of di-acylated MEL producers in the genus. These strains produced MELs including the mono-acylated one at a rate of 20–25%. From these results, the new MEL is likely to have great potential for use in oil-in-water-type emulsifiers and washing detergents because of its higher water solubility compared to conventional MELs and will thus contribute to facilitating a broad range of applications for the environmentally advanced surfactants.     

<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.     

<Emphasis Type="Italic">O</Emphasis>-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac₂-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac₂-GPs_ALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac₂-GPs on PBMC_ALL with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA⁻) revealed decreased nitric oxide production as compared to 9-O-AcSA⁺ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-O-AcSA⁻ cells as compared to 9-O-AcSA⁺ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac₂-GPs are also capable of inducing disease-specific anti-Neu5,9Ac₂-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac₂-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac₂-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.     

α-(4-O-Methyl)-d-glucuronidase activity produced by the rumen anaerobic fungusPiromonas communis: A study of selected properties

The rumen anaerobic fungusPiromonas communis, unlike the rumen anaerobic fungiNeocallimastix frontalis andNeocallimastix patriciarum, produced extracellular α-(4-O-methyl)-d-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acidO-α-(4-O-methyl-d-glucopyran-osyluronic acid)-(1 → 2)-d-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid (1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₂) and the aldotetraouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid)-(1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl₃) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylooligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly,p-nitrophenyl-α-d-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the α-(4-O-methyl)-d-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The α-(4-O-methyl)-d-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50°C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100–300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.     

Synthesis and biological properties of pyrimidine 4′-fluoronucleosides and 4′-fluorouridine 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphate

4′-Fluoro-2′,3′-O-isopropylidenecytidine was synthesized by the treatment of 5′-O-acetyl-4′-fluoro-2′,3′-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. The interaction of 4′-fluoro-2′,3′-O-isopropylidenecytidine with hydroxylamine resulted in 4′-fluoro-2′,3′-O-isopropylidene-5′-O-acetyl-N ⁴-hydroxycytidine. The removal of the 2′,3′-O-isopropylidene groups led to acetyl derivatives of 4′-fluorouridine, 4′-fluorocytidine, and 4′-fluoro-N ⁴-hydroxycytidine. 4′-Fluorouridine 5′-O-triphosphate was obtained in three steps starting from 4′-fluoro-2′,3′-O-isopropylideneuridine. 4′-Fluorouridine 5′-O-triphosphate was shown to be an effective inhibitor of HCV RNA-dependent RNA polymerase and a substrate for the NTPase reaction catalyzed by the HCV NS3 protein, the hydrolysis rate being similar to that of ATP. It could also activate a helicase reaction with an efficacy of only threefold lower than that for ATP.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Regioselective deglycosylation of onion quercetin glucosides by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Bioconversion of quercetin glucosides using four generally recognized as safe (GRAS) organisms (Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae) was evaluated by measuring changes in the levels of quercetin compounds of onion. Of the four organisms, S. cerevisiae increased the content of quercetin-3-O-β-d-glucoside (III; isoquercitrin) and quercetin (IV), whereas decreasing quercetin-3,4′-O-β-d-glucoside (I) and quercetin-4′-O-β-d-glucoside (II). Also, S. cerevisiae converted authentic compound I to III, and II to IV, respectively. These results suggest that S. cerevisiae can be used to increase the levels of isoquercitrin (III), the most bioavailable quercetin compound in onion.