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A new cycloartane glycoside was isolated from the dried whole herbs of Camptosorus sibiricus Rupr By means of chemical (hydrolysis) and spectroscopic methods (IR, 1D, and 2D NMR, ESI-MS), the structure was established as 3beta, 7beta, 24beta, 25, 30-pentahydroxycycloartane-3-omicron-beta-D-glucopyranoside-24-omicron-beta-D-glucopyranoside (1).
Two new phenylpropanoid glycosides, 1-beta-D-glucopyranosyl-2,6-dimethoxy-4-propenylphenol (1) and 1-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-2,6-dimethoxy-4-propenylphenol (2) were isolated from the stem bark of Acanthopanax trifoliatus along with four known compounds (3-6). Their structures were established on the basis of spectral and chemical evidences.
The mass spectrometric development of an enzymatic assay resulting in enzymatic activity, its reaction pathway and its dissociation constant are described for the first time within a single experiment. The method combines the performance of a mass spectrometry-compatible enzyme assay with the direct detection of specific enzyme complexes using hen egg white lysozyme as a model. The continuous liquid-flow technique applied, when hyphenated with electrospray ionization (ESI)-time-of-flight (ToF)-mass spectrometry (MS), permitted the simultaneous detection of several substances involved in product screening as well as the direct observation of dissociation constants. Dissociation constants for the product inhibitor N, N', N''-triacetylchitotriose were calculated using a Scatchard plot (12 x 10(-6) M) and the law of mass action (18-24 x 10(-6) M), and these are in good agreement with constants obtained in earlier mass spectrometric (6-18 x 10(-6) M) or spectroscopic (6-8 x 10(-6) M) studies. Finally, the enzymatic hydrolysis of glycosidic substrate was monitored by ESI-ToF-MS in the presence of various inhibitors, thus leading to decreased activities in terms of their enzyme affinities. The associated inhibitor-enzyme complexes could be detected for up to lower micromolar K( d ) values.
OBJECTIVE: Cardiac glycosides exert their inotropic effect by increasing intracellular calcium. Increased intracellular calcium is a key event in platelet aggregation. In aggregometer studies, digitalis has been found to augment platelet agonist responses. A prothrombotic effect of digitalis might be concealed since heart failure and atrial fibrillation per se predispose to thromboembolism. The present study investigates the effects of digitoxin on platelet function in healthy volunteers. METHODS: Twenty healthy, non-smoking volunteers were randomised to receive digitoxin ( n = 10, 0.6 mg day 1, 0.4 mg day 2, then 0.1 mg daily) or placebo ( n = 10) for 10 days. Platelet function was then analysed ex vivo using three-colour whole-blood-flow cytometry, both in non-stimulated mode and after agonist stimulation with 0.1 micromol/l adenosine diphosphate (ADP), 10 micromol/l ADP and 5.0 micromol/l epinephrine (final concentrations). Expression of activated fibrinogen receptor, von Willebrand's factor receptor and P-selectin, formation of platelet-platelet and platelet-leukocyte aggregates and particle size were examined. RESULTS: No significant difference between the placebo and the digitoxin group (digitoxin levels 17-42 nmol/l) was found, neither on a global level nor for any isolated parameter. CONCLUSIONS: Theory and in vitro data suggest that digitoxin treatment could activate platelets. No evidence for this was found in healthy volunteers. This observation is strengthened by the unequivocal results for all parameters measured. However, thrombosis-prone patients with heart failure and/or atrial fibrillation may respond differently to digitalis therapy.
Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na(+)/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K(0.5) 0.06 mm) and 2-naphthylglucose with lower affinity (K(0.5) 0. 5 mm) than alpha-methyl-d-glucopyranoside (alpha MDG, 0.2 mm). Both were poorly transported (maximal velocities, I(max), 14% and 8% of alpha MDG). Other compounds were inhibitors (K(i)s 1-13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than alpha MDG (K(0.5)s 0.9, 0.2 and 2.5 mm and relative I(max)s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I(max)s (130 and 120%) and comparable K(0. 5)s (8 and 1 mm). Increased affinity of indican relative to alphaMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value.
When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for beta-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1->6)-beta-D-Glcp-(1->3)-beta-D-Glcp-(1->3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1->4)-beta-D-Glcp-(1->3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes beta-D-Glcp-(1->3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.
The rumen anaerobic fungus Caecomyces communis was grown in a fermentor in Lowe medium. We studied four polysaccharide hydrolases and three glycoside hydrolases at early and final stages. We found a difference in cell association for these enzymes depending on the developmental stage. The endocellulase and beta-D-fucosidase were early synthesized, and their activities decreased at the end of the developmental cycle. On the contrary, the beta-D-glucosidase, beta-D-xylosidase and xylanase activities increased during the cycle. The avicelase and the CM-cellulase activities linked with thalli increased, whereas the extracellular activities of these enzymes decreased.
1,2,3,4-tetra-O-acetyl-6-ethyladipate-beta- D-glucopyranose (TOGE), a glycoside derivative of adipic acid monoethyl ester and 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose, was synthesized and the resistance-inducing activity shown by TOGE-1 (TOGE plus furfurylamine) and TOGE-2 (TOGE plus 1,3-diaminepropane) was assayed. TOGE-1 and TOGE-2 protected tomato plants against two different fungal pathogens Phytophthora citrophthora and Alternaria solani. Foliar treatments at very low concentrations (2.5 mg l(-1) TOGE, 0.5 mg l(-1) amine) clearly reduced the disease incidence for both pathogens. TOGE-2 application was the most effective on intact plants as well as on detached leaves, reducing fungal growth by more than 46% with respect to control plants. On the other hand TOGE-1 treatment reduced fungal advance by 21%. These results demonstrate a high protective effect against fungal infections for both chemicals. A possible direct antimicrobial effect was discounted due to the weak activity observed in vitro against these pathogens at the low concentrations used in plants. TOGE-2 treatment clearly activates resistance against both pathogens and improves the protective effect previously shown by FGA mixture (adipic acid monoethyl ester, 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose and furfurylamine) [V. Flors et al. (2001) J Agric Food Chem 49:2569-2575]. The obtained results indicate that TOGE-1 and TOGE-2 act as resistance inducers. Although their mode of action is still unknown, pre-challenge studies have demonstrated the induction of the phenylpropanoid pathway and antioxidant activities. Both chemicals have demonstrated a beneficial effect on plant development, increasing chlorophyll and protein contents, photosynthetic rate and water-use efficiency. The improvement of plant growth and development produced by these treatments suggests crop tolerance to these chemicals, although effective formulations that are safe to humans must be developed before this technology can be used commercially.
The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.
Salix sericea (Marsh.) (Salicaceae) seedlings were used to investigate phytochemical induction of phenolic glycosides following beetle herbivory. Seven-week-old full-sibling seedlings were subjected to one of three damage treatments: Plagiodera versicolora adults, P. versicolora larvae, or Calligrapha multipunctata bigsbyana adults. Salicylate concentrations were measured locally (within damaged leaves) and systemically (above and below damaged leaves) 4 d later. Herbivory caused differential salicylate induction; 2'-cinnamoylsalicortin was induced, whereas salicortin was not. The induction of 2'-cinnamoylsalicortin was not specific with regard to the species or developmental stage of beetle tested but did vary with leaf age: induction occurred in the younger undamaged leaves but not in the damaged leaves or in the older undamaged leaves. The amount of leaf area consumed had no detectable effect on induction, indicating an "all-or-none" response triggered by even small amounts of herbivory. Locally, herbivory caused a decrease in salicortin concentrations, probably because of degradation within the damaged leaves. These results suggest a specific but generalized induced response to these leaf-feeding beetles.
In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.
Carotenoid glycosides and isoprenoid glycolipids are of great interest, especially for the medicinal, pharmaceutical, food, cosmetic, flavor, and fragrance industries. These biologically active natural surfactants have good prospects for the future chemical preparation of compounds useful as antimicrobial, antibacterial, and antitumor agents, or in industry. More than 300 unusual natural surfactants are described in this review article, including their chemical structures and biological activities.
FA amide glycosides are of great interest, especially for the medicinal and pharmaceutical industries. These biologically active natural surfactants are good prospects for future chemical preparation of compounds useful as antibiotics, anticancer agents, or for industry. More than 200 unusual and interesting natural surfactants, including their chemical structures and biological activities, are described in this review article.
A rapid extraction method followed by high-performance liquid chromatographic assay was developed for the quantitative determination of the cardioactive glycosides of Digitalis lanata. The leaf samples were extracted with water or aqueous alcohols. The simple extraction method gives a better yield than the methods described previously. Lanatoside C and its metabolites have been separated on a reversed-phase column with various mixtures of acetonitrile, methanol, and water as mobile phases for isocratic elution. Extraction and quantitative determination of lanatoside C and digoxin from a leaf sample require not more than 30 min.
The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.
The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.
In this study the pharmacological effects of seven semisynthetic cardenolides have been investigated and compared with those of 11 natural cardiac glycosides. These compounds are of different potency on electrically driven isolated guinea-pig atria. Their concentration response curves showed different slopes, which could be an indication of varying therapeutic range. The pharmacodynamics of these compounds on isolated guinea-pig atria are in good correlation with the data obtained from binding studies on guinea-pig ventricular homogenate as well as that obtained from comparative experiments on Na+,K+-ATPase activity inhibition.
The cross-resistance patterns of two different types of mutants of HeLa cells selected for resistance to the digoxin analog SC4453 (SCR mutants) in which the Na+/K+-ATPase is affected [A. Chopra and R. S. Gupta, J. biol. Chem. 261, 2034 (1986)], and towards numerous cardiac glycosides (CGs) and genins, were examined. One type of SCR mutant (designated as group C) was highly resistant to all CGs and genins investigated. In contrast, the other type of SCR mutant (group D) showed a high degree of cross-resistance towards selected CG derivatives (viz. digoxin, SC4453, digoxigenin, lanatoside C, alpha- and beta-methyldigoxin, dihydrodigoxin, alpha- and beta-acetyldigoxin, alpha,beta-diacetyldigoxin), all of which contained a free 12 beta-OH group in the steroid structure. Slight cross-resistance of the group D mutants was also observed for other compounds (viz. ouabain, ouabagenin, dihydroouabain) that contain a free 11 alpha-OH group in the molecule. However, these mutants exhibited no cross-resistance to other CG derivatives, which either lacked the above groups (viz. digitoxin, digitoxigenin, dihydrodigitoxin, digitoxigenin mono- and bisdigitoside, nerifolin, gitoxigenin, gitoxin, 16-acetylgitoxin, lanatosides A and B, cymarin, convallatoxin, oleandrin, strophanthidin, actodigin and bufalin) or in which the 12 beta-OH group was acetylated (viz. as in the case of 12-acetyldigoxin). Since the 12 beta-OH group is not required for CG-like activity, to account for these observations it is suggested that the genetic lesion in the group D mutant leads to the creation of a new binding site in the digitalis receptor, which specifically interacts with the 12 beta-OH group (the site presumably also interacts weakly with the 11 alpha-OH group) and either prevents or distorts the binding of the compounds to the drug binding site on the receptor. Further investigations with the different classes of CG-resistant mutants at the molecular level should prove very useful in identifying the drug receptor site and in understanding how these drugs interact with it.