Cyclopentenylglycine,a precursor of deidaclin in Turnera ulmifolia

When [2-¹⁴C]cyclopentenylglycine was synthesized and fed to seedlings of Turnera ulmifolia, the label was incorporated into the nitrile group of the cyanogenic glycoside deidaclin. The amino acid cyclopentenylglycine was also found to occur naturally in Turnera ulmifolia. These findings indicate that cyclopentenyl cyanogenic glycosides are synthesized from the corresponding amino acids by the same pathway utilized in the biosynthesis of other cyanogenic glycosides.     

Electrophysiological studies of gustation in lepidopterous larvae

Summary The electrophysiological responses of the maxillary gustatory sensilla of ten species of lepidopterous larvae to sugars, polyhydric alcohols, amino acids, organic acids, glycosides, and sterols were tested. Of the caterpillars examined three were monophagous (Calpodes ethlius, Ceratomia catalpae, Danaus plexippus), three were oligophagous (Pieris rapae, Papilio polyxenes, Malacosoma americana), and four were polyphagous (Helioihis zea, Estigmene acrea, Isia isabella, Porthetria dispar). The sensilla of all species are structurally similar to the extent of having four chemosensory bipolar neurons in each peg.It is not possible to account for the wide range of responsiveness to diverse compounds in terms of narrowly specific receptors characterized as water, sugar, salt, inositol, or amino acid receptors. Bach receptive cell was found to have only a limited degree of specificity. Not all amino acids stimulated the same cell. Some amino acids stimulated only one; some stimulated two; some inhibited all cells; some inhibited one while stimulating another. Differential sensitivity to amino acids varied from species to species and even between the two sensilla in an individual.The diversity of relationships found with amino acids existed also with respect to acids and glycosides. Ascorbic and hydrochloric acids at equivalent pH values each stimulated a different cell. Glycosides stimulated one or two cells depending on the species and sensillum.Synergism occurred nearly as often as inhibition. Among the instances were synergistic reactions between sucrose and inositol, proline and inositol, sucrose and sinigrin, glycine and sucrose. The occurrence of the phenomenon was specific to certain species and sensilla.Results point to the conclusion that the basis of difference in sensitivity to different compounds from species to species and from receptor to receptor reflects variations in the molecular structure of the receptor substrate. Whatever the mechanisms involved it is clear that caterpillars are able to detect not only compounds that may be of nutritive value but also non-nutritive compounds some of which are secondary plant substances. When the total taste sensitivities of the various species of caterpillars are compared, there is no obvious difference between polyphagous, oligophagous, and monophagous species.This work was supported by National Science Foundation Grant GB 1472.     

Molecular basis for the insensitivity of the Monarch (Danaus plexippus) to cardiac glycosides.

The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouabain inhibits the Na+,K(+)-ATPase by binding to its alpha-subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Freeze-drying and analysis of plant and other biological material

The freeze-drying of biological material, which is to be quantitatively analyzed (micro-amount level) for compounds of low or intermediate molecular weight, should be either omitted or handled under strict control. This is because compounds such as amino acids, sugars, flavonoids, glycosides, coenzymes, peptides, etc., might be removed from concentrates and (or) the ground biological material by the high vacuum.     

Studies on Cassava, Manihot utilissima. II. Biosynthesis of Asparagine-14C from 14C-labelled Hydrogen Cyanide and its Relations with Cyanogenesis

Seeds and seedlings of Manihot utilissima were analysed for cyanogenic glycosides und free amino acids, with special reference to valine and isoleucine which serve as precursors of the aglycone moieties of linamarin and lotaustralin. Seeds contained traces of valine and isoleucine but no glycosides, whereas seedlings contained high concentrations of these amino acids and glycosides. Illumination of seedlings led to a steep increase in the concentration of glycosides followed by a decrease without excretion of detectable HCN. Seeds accumulated asparagine, while seedlings accumulated both asparagine and glutamine in the storage and transport of nitrogen. Seedlings incorporated 13.2 per cent of label from valine-¹⁴C(U) and 2.4 per cent of label from isoleucine-¹⁴C(U)into linamarin and lotaustralin, respectively. In both cases, appreciable amounts of label were also incorporated into asparagine. 49 per cent of label from H¹⁴CN was incorporated inio asparagine in which ca. 98 per cent of total radioactivity was located in the amide-carbon atom. The different patterns of labelling which occurred during the assimilation of H¹⁴CN and ¹⁴CO₂ showed that cyanide metabolism did not proceed via CO₂, and that M. utilissima contains an efficient enzyme-system which catalyses the conversion on high concentrations of HCN into asparagine, which subsequently enters different metabolic pools involved with respiration, protein and carbohydrate syntheses. Cyanogenesis in M. utilissima appears lo be directly influenced by available pools of valine and isoleucine, and the metabolism of HCN released from linamarin and lotaustralin by the action of linamarase may be directly related to respiratory and synthetic processes by way of the incorporation of HCN as a unit into asparagine.     

Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.     

Induction of apoptosis by alkaloids, non-protein amino acids, and cardiac glycosides in human promyelotic HL-60 cells

The induction of apoptosis by 66 alkaloids of the quinoline, quinolizidine, pyrrolizidine, isoquinoline, indole, terpene, tropane, steroid, purine, and piperidine type, of 9 cardiac glycosides, 11 non-protein amino acids and 10 further secondary metabolites was assayed in HL-60 cell cultures and measured by quantification of the subdiploid DNA content by flow cytometry, detection of DNA fragmentation by gel electrophoresis, and cell morphology. Several alkaloids of the isoquinoline, quinoline, and indole type were active, whereas quinol-izidine, tropane, pyrrolizidine, terpene and piperdine alkaloids were mostly inactive. The proapoptotic alkaloids can be characterized by their property to inhibit protein biosynthesis and their intercalation into DNA at the same time, or by their inhibition of microtubule formation. All cardiac glycosides, which are both membrane detergents and Na+,K+-ATPase inhibitors, are potent apoptosis inducers. Also proapoptotic were a few non-protein amino acids, podophyllotoxin and the flavonoid quercetin.     

A unique pantoyllactone glycoside system is activated in rice seedlings developing aerobically in the dark

Rice ( Oryza sativa L.) seedlings developing aerobically in the dark accumulated in the expanding coleoptile millimolar amounts of pantoyllactone glucoside (PLG) together with sugars and amino acids. Following leaf emergence and seedling development, the transformation of PLG into pantoyllactone primeveroside (PLP) was initiated inside the coleoptile, whose senescence was suspended in spite of the exhaustion of seed reserves and cessation of shoot growth. PLG, PLP and most of the sugars and amino acids remained inside the coleoptile. Light administration triggered shoot greening and coleoptile senescence that was accompanied by the conversion of PLG into the shoot-translocatable species PLP and by the utilization of the coleoptile-stored metabolites. Light-induced retrieval of amino acids and sugars from the coleoptile was 90% complete within 24 h while translocation of pantoyllactone glycosides started only 24–48 h after light administration. Conversely, no accumulation of PLG and PLP was detected in seedlings germinated and grown either aerobically in the light or in the dark under hypoxic conditions. The possible significance of the activation of the PLG-PLP system under specific environmental conditions is discussed.     

Carbohydrate bond to the hydroxyaromatic amino acids in actinoidins A and B

The method of exhaustive methylation with diasomethane was used as applied for actinoidines A and B and their glycosides for elucidation of the problem of connection between the carbohydrates and oxyaromatic fragments of the antibiotics followed by analysis of the acid hydrolyzates of the methoxy derivatives. It was found that the disaccarhide branch, i.e. 2-O-(L-acozaminyl)-D-glucose was connected in actinoidines A and B through the phenol group of the triaminotricarbonic fragment V, while the residue of D-mannose was connected through one of the phenol groups of the actinoidinic amino acid. The other two phenolic groups of the actinoidinic amino acid, as well as the phenolic groups of the N-end amino acids did not participate ion formation of the glycoside bonds. Actinosamine was probably connected through the alcohol group of the aglycone.     

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.     

Identification of tyrosinase inhibitors from Marrubium velutinum and Marrubium cylleneum

Tyrosinase is a key enzyme in the production of melanins in plants and animals. Forty-five secondary metabolites isolated from Marrubium velutinum and Marrubium cylleneum belonging to the classes of flavonoids, phenylethanoid glycosides, phenolic acids and lignan glycosides were screened for their inhibitory activity against mushroom tyrosinase. Flavonoids and phenylethanoid glycosides showed moderate inhibitory activity, while phenolic acids were less active than phenylethanoid glycosides, suggesting that both phenolic groups are important for the activity.     

Purification and characterization of an agglutinin from mucus of the snail Achatina fulica

The mucus of the snail Achatina fulica shows the presence of an agglutinin that nonspecifically agglutinates human erythrocytes. The agglutinin has been purified by affinity chromatography using Sepharose 4B-hog gastric mucin as the affinity matrix. Homogeneity was checked by polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and gel filtration. The agglutinin is a glycoprotein of native molecular weight 70,000. The isoelectric point of the protein was found to be 8.0. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 32% of the total amino acid residues. The agglutinin has 10% carbohydrate (wt/wt) and the most abundant sugar is N-acetylglucosamine. The cd spectra of the agglutinin show the presence of random coil conformation. The inhibition of hemagglutination data indicates that the agglutinin is specific for beta glycosides of D-Gal and D-GalNAc.     

笃斯越桔化学成分及其功能活性的研究进展

笃斯越桔(Vaccinium uliginosum)因其富含花青苷等生物活性成分而备受关注。迄今为止, 从笃斯越桔果实中已鉴定出24种花青苷、32种黄酮醇苷、24种有机酸、28种芳香成分及少量儿茶素及其低聚物。同时, 笃斯越桔还含有丰富的矿质元素、维生素及氨基酸等营养成分。大量的体内和体外实验结果表明, 笃斯越桔具有强大的抗氧化、抗衰老、抗炎、抗癌、提高记忆力、预防心血管疾病和保护视力等生理活性。该文介绍了笃斯越桔资源分布情况及其遗传背景, 综述了其果实和叶片中的主要功能成分、营养物质及芳香成分的组成和含量, 讨论了遗传和环境等因素对其活性物质合成和积累的影响, 以期为其新品种选育及综合利用提供参考依据。     

Xylanase treatment of plant cells induces glycosylation and fatty acylation of phytosterols

Treatment of tobacco suspension cells ( Nicotiana tabacum cv. KY 14) with a purified β -1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)⁻¹] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu²⁺ or Ag⁺ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β -sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.     

N-acetylglucosaminides. A new type of bile acid conjugate in man

Bile acids were extracted from human urine and were separated into groups of nonamidated and glycine- and taurine-conjugated compounds. Each group was subfractionated in a reversed-phase high performance liquid chromatography system, and the fractions were analyzed by negative ion fast atom bombardment mass spectrometry and also by gas chromatography-mass spectrometry after enzymatic removal of glycine and taurine moieties. The major glycosides of the non-amidated bile acids were more polar than reference bile acid glucosides and gave quasimolecular ions at m/z 592, 594, and 610 consistent with N-acetylglucosaminides of unsaturated dihydroxy and saturated di- and trihydroxy bile acids. Gas chromatography-mass spectrometry analyses of methyl ester trimethylsilyl ether derivatives showed fragments typical for N-acetylglucosaminides (m/z 173 and 186) in addition to those also given by glucosides (m/z 204 and 217). The N-acetylglucosaminides were inert toward alpha- and beta-glucosidase but were cleaved completely with N-acetylglucosaminidase. The released sugar moiety was identified as N-acetylglucosamine. One of the liberated bile acids was identified as ursodeoxycholic acid. The other acids were not identical to any known primary or secondary bile acid in humans. Fast atom bombardment mass spectrometry analyses of the glycine-and taurine-conjugated bile acid glycosides only showed ions consistent with the presence of glucosides (m/z 626 and 676). These compounds were sensitive only toward beta-glucosidase which liberated a trihydroxy bile acid as the major compound. Based on the recover of 13C- and 14C-labeled chenodeoxycholic acid glucoside added as internal standard, the daily excretion of nonamidated bile acid glycosides was estimated to be about 137 micrograms or 0.29 mumol, N-acetylglucosaminides constituting about 90%. The daily excretion of the glucosides of amidated bile acids was about 150 micrograms or 0.25 mumol, glycine conjugates constituting about 90%.     

Far-ultraviolet circular dichroism and uronic acid components of anticoagulant deca-, dodeca-, tetradeca-, and octadecasaccharide heparin fractions

The therapeutic anticoagulant action of heparin is mediated by the ability of a multifunctional octadecasaccharide region of the molecule to bind to and differentially alter the conformational integrity of antithrombin, and the sugar sequence of the primary binding domain is known. Low ultraviolet circular dichroism spectroscopy of heparin-derived anticoagulant octa-, deca-, dodeca-, tetradeca-, and octadecasaccharides has been useful in elucidating the nature of the sugars that are contained within the second functional domain of the octadecasaccharide. The difference between the spectra of the molar ellipticity of the above sequential oligosaccharides was taken to be the CD spectrum of the corresponding additional disaccharide unit(s). Optical models of the component disaccharides of heparin were derived from CD spectra of heparins having a high degree of sulfation, synthetic glycosides of N-acetylglucosamine, and glycosides of component uronic acids. These were sufficiently distinct in magnitude and spectral position to warrant interpretation of the experimental difference CD spectra. The uronic acids of the disaccharides between deca- and octamer, dodeca- and decamer, and tetradeca- and dodecamer were thereby ascribed to sulfated iduronate, unsulfated iduronate, and glucuronate residues, respectively, while those of the tetrasaccharide between the octadeca- and tetradecasaccharide were tentatively assigned to sulfated iduronate moieties. Interpretation of the difference CD spectra on the basis of the optical models was less certain in regard to the amino sugar components. It appears that the amino sugar derivative between the dodeca- and decamer was N-acetylglucosamine, while the other disaccharides of the octa- to octadecasaccharide probably contained the N-sulfated derivative. A speculative disaccharide sequence drawn from these data indicates that relatively less strongly anionic disaccharides, having nonsulfated uronic acid moieties and N-acetylglucosamine, were flanked by trisulfated disaccharide units, constituting a structural element similar to that which contains the primary binding domain of the anticoagulant.     

Sterols in relation to ageing of seeds of Helianthus annuus and Cicer arietinum

Under accelerated ageing at high relative humidity and high temperature for 4 days germination and membrane permeability remained unaffected both in sunflower and chick pea seeds. However, the steryl glycoside concentration in the pooled leachate increased progressively with ageing. Total sterols, as well as steryl glycosides and free sterols of the seeds, increased with a concomitant decline in steryl esters under accelerated ageing. Pretreatment with the sterol biosynthesis inhibitor SK & F 7997A₃ prevented the increase of total sterols under accelerated ageing conditions but there were increases in the amounts of steryl glycosides and free sterols and a decrease in steryl ester after such treatment, therefore, indicating interconversions of the various sterol types. Accelerated ageing also caused increases in free amino acids and soluble carbohydrate. Low relative humidity-high temperature and high relative humidity-low temperature failed to produce such effects.     

Synthesis of glycuronamides of amino acids,constituents of microbial polysaccharides and their conversion into neoglycoconjugates of copolymer type

Glycopyranosiduronic acids, amidically linked to amino acids (alanine, serine, threonine, and lysine) were prepared.O-tert-Butyl andN-tert-butyloxycarbonyl protected amino acidtert-butyl esters were used in ethyl 2-ethoxy-1,2-dihydroquinoline-1-carboxylate promoted condensation with 2-azidoethyl glycosides of glucuronic and galacturonic acid. Reduction of the azido-function followed byN-acryloylation and removal of blocking groups with trifluoroacetic acid gave the target monomers. These were converted into neoglycoconjugates of copolymer type, potentially useful for immunochemical studies.On leave from the Indian Institute of Chemical Technology, Hyderabad, India.     

Characterization of three terpenoid glycosyltransferase genes in 'Valencia' sweet orange (Citrus sinensis L. Osbeck)

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in 'Valencia' sweet orange (Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730-874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.