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PubMed1974

Cleavage of the glycosidic linkage of pyrimidine ribonucleosides by the bisulfite-oxygen system.

When a solution containing 2 mM uridine, 20 mM sodium bisulfite, 0.1 mM MnCl(2), and 100 mM sodium phosphate buffer of pH 7.0 was incubated aerobically at 37 degrees or 0 degrees , partial cleavage of the glycosidic linkage of uridine took place. About 20% of the uridine was converted to uracil by the incubation for 4 hrs. Cytosine was produced from cytidine by similar treatment with bisulfite. These reactions were caused by free radicals generated by Mn(2+)-catalyzed autoxidation of bisulfite. Glycosidic bond cleavage by the bisulfite-oxygen system was not detected for adenosine, AMP, guanosine, GMP, thymidine, TMP, deoxyuridine, dCMP, dAMP, and dGMP. When poly(U) and poly(C) were treated with 20 mM sodium bisulfite in the same manner, chain fission of the polymer occurred as judged by the elution-pattern change in gel filtration through Sephadex columns. No change in the elution pattern was observed for bisulfite-treated poly(A), poly(U). poly(A) or tRNA.

Binding Sites↗

PubMed1996

Synthesis and characterization of 8-methoxy-2'- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2'-deoxyadenosine within DNA.

The synthesis of 8-methoxy-2'-deoxyadenosine (moA) protected at N6 as an N,N-dimethylformamidine derivative and incorporation of the modified nucleoside into oligodeoxynucleotides via the phosphoramidite method are described. UV thermal denaturation studies were conducted on duplexes containing moA:G, moA:C and moA:T base pairs to determine the thermodynamic stability of duplexes containing moA relative to their adenosine (A)-containing counterparts. In the case of moA:G base pairs the effect of moA substitution is sequence dependent. In A:G mismatch-containing sequences, which have been shown by structural characterization to have a syn conformational preference at the glycosidic bond of A, moA substitution results in stabilization of the duplex. In contrast, in sequences where the A in the A:G mismatch has been shown to prefer the anti conformation moA substitution is destabilizing to the duplex. Thus moA may be a useful probe for investigating the conformational preferences of the N-glycosidic bond of adenosine within DNA. In addition, moA nucleoside is more resistant to acid-catalyzed depurination than previously described 8-bromo-2'-deoxyadenosine, allowing for facile incorporation into oligonucleotides via automated solid phase DNA synthesis.

Base Sequence↗

PubMed2001

Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells.

The saponin digitonin, the aglycone digitoxigenin and five cardiac glycosides were evaluated for cytotoxicity using primary cultures of tumor cells from patients and a human cell line panel (representing different cytotoxic drug-resistance patterns). Of these seven compounds, proscillaridin A was the most potent (IC(50): 6.4--76 nM), followed by digitoxin, and then ouabain, digoxin, lanatoside C, digitoxigenin and digitonin. Correlation analysis of the log IC(50) values for the cell lines in the panel showed that compound cytotoxicity was only slightly influenced by resistance mechanisms that involved P-glycoprotein, topoisomerase II, multidrug resistance-associated protein and glutathione-mediated drug resistance. Digitoxin and digoxin expressed selective toxicity against solid tumor cells from patients, while proscillaridin A expressed no selective toxicity against either solid or hematological tumor cells. The results revealed marked differences in cytotoxicity between the cardiac glycosides, both in potency and selectivity, and modes of action for cytotoxicity that differ from that of commonly used anticancer drugs.

Antineoplastic Agents↗

PubMedDate not supplied

Deleterious synergism of a cardiac glycoside and sodium diatrizoate.

Studies were performed in mice to determine if the cardiac glycoside, Strophanthin-K, and the contrast medium, sodium diatrizoate, interact synergistically to produce death. Intravenous injections of lethal and near lethal doses of the two agents produced a significantly greater mortality than the individual agents alone. Low or near clinical doses of Strophanthin-K, when given with doses of diatrizoate in the lethal range, produced mortalities significantly greater than did the diatrizoate alone. Similarly, low or near clinical doses of diatrizoate given with doses of Strophanthin-K in the lethal range produced mortalities significantly greater than for the Strophanthin-K alone. Isotonic or hypertonic saline, when substituted for diatrizoate or Strophanthin-K did not produce synergistic increases in mortality. Thus neither the injection volume, nor agent hypertonicity or ionic strength, seem to be the primary factors in the synergism to produce death. The diatrizoate anion appears to be an important factor. Until more information is available from other animal models it appears that patients receiving cardiac glycoside should be considered to have a higher than normal risk of serious reactions to contrast media in intravenous urography.

Animals↗

PubMed2003

Endogenous glycosides in critically ill patients.

OBJECTIVE: To determine the incidence of critically ill patients displaying endogenous digitalis-like-immunoreactive substances (DLIS) and to examine the relationship of these hormones to routine laboratory variables, the underlying disease, myocardial function, hemodynamic status, severity of illness, systemic inflammation, and mortality rate. DESIGN: Sera of 401 consecutive critically ill patients, not treated with cardiac glycosides, were analyzed for DLIS (digitoxin and digoxin, TDx; Abbott Diagnostics, North Chicago, IL) and endogenous ouabain. Normal values of endogenous ouabain were determined in 62 healthy volunteers. We measured pro- and anti-inflammatory mediators (L-selectin, tumor necrosis factor-alpha, interleukin-1beta, interleukin-2, interleukin-6, interleukin-10), C-reactive protein, and serum amyloid A protein as well as patients' Acute Physiology and Chronic Health Evaluation II and Goris scores. In a subgroup of patients with a pulmonary artery catheter (n = 95), we determined cardiac output, pulmonary artery occlusion pressure, systemic and pulmonary vascular resistance, left ventricular stroke volume, and right and left stroke work. SETTING: Two surgical intensive care units of an university hospital. SUBJECTS: Sera of 401 consecutive critically ill patients. INTERVENTIONS: Blood sampling. MEASUREMENTS AND MAIN RESULTS: Of the 401 patients tested, 343 had nonmeasurable DLIS concentrations (DLIS-negative), and 58 (14.5%) had positive digoxin (n = 18) or digitoxin (n = 34) concentrations (DLIS-positive) or were positive in both tests (n = 6). Mean endogenous ouabain concentrations were nine-fold increased in DLIS-positive (3.59 +/- 1.43 nmol/L) and three-fold increased in DLIS-negative (1.34 +/-.81 nmol/L) patients compared with controls (0.38 +/- 0.31 nmol/L). DLIS and ouabain concentrations closely correlated with the Acute Physiology and Chronic Health Evaluation II and Goris score and were associated with increased concentrations of transaminases, bilirubin, aldosterone, cortisol, serum creatinine, fractional sodium excretion, proinflammatory mediators, C-reactive protein, and serum amyloid A (p <or=.009). The hospital mortality rates of DLIS-positive and DLIS-negative patients were 12% and 3.2%, respectively, and for patients with ouabain concentrations above and below 2 nmol/L 38.6% and 0.6%, respectively. In DLIS-positive patients with pulmonary artery catheter (n = 23), cardiac output, stroke volume, and left ventricular stroke work were decreased, and pulmonary artery occlusion pressure and central venous pressure were increased (p <or=.009). CONCLUSIONS: Different types of endogenous glycosides including endogenous ouabain are elevated in a significant proportion of critically ill patients. The occurrence of these substances is associated with increased morbidity and hospital mortality rates, possibly due to systemic inflammatory processes. DLIS but not endogenous ouabain concentrations were found to be related to left ventricular function.

APACHE↗

PubMed1993

The Role of Flavonol Glycosides and Carotenoids in Protecting Soybean from Ultraviolet-B Damage.

The increase in ultraviolet-B (UV-B; 0.290-0.320 [mu]m) radiation received by plants due to stratospheric ozone depletion heightens the importance of understanding UV-B tolerance. Photosynthetic tissue is believed to be protected from UV-B radiation by UV-B-absorbing compounds (e.g. flavonoids). Although synthesis of flavonoids is induced by UV-B radiation, its protective role on photosynthetic pigments has not been clearly demonstrated. This results in part from the design of UV-B experiments in which experimental UV-A irradiance has not been carefully controlled, since blue/UV-A radiation is involved in the biosynthesis of the photosynthetic pigments. The relationship of flavonoids to photosynthetic performance, photosynthetic pigments, and growth measures was examined in an experiment where UV-A control groups were included at two biologically effective daily UV-B irradiances, 14.1 and 10.7 kJ m-2. Normal, chlorophyll-deficient, and flavonoid-deficient pigment isolines of two soybean (Glycine max) cultivars that produced different flavonol glycosides (Harosoy produced kaempferol, Clark produced quercetin and kaempferol) were examined. Plants with higher levels of total flavonoids, not specific flavonol glycosides, were more UV-B tolerant as determined by growth, pigment, and gas-exchange variables. Regression analyses indicated no direct relationship between photosynthesis and leaf levels of UV-B-absorbing compounds. UV-B radiation increased photosynthetic pigment content, along with UV-B-absorbing compounds, but only the former (especially carotenoids) was related to total biomass (r2 = 0.61, linear) and to photosynthetic efficiency (negative, exponential relationship, r2 = 0.82). A reduction in photosynthesis was associated primarily with a stomatal limitation rather than photosystem II damage. This study suggests that both carotenoids and flavonoids may be involved in plant UV-B photoprotection, but only carotenoids are directly linked to photoprotection of photosynthetic function. These results additionally show the importance of UV-A control in UV-B experiments conducted using artificial lamps and filters.

Journal Article↗

PubMed1980

Glycosidic Enzyme Activity in Pea Tissue and Pea-Fusarium solani Interactions.

Membrane barriers which prevent direct contact between Fusarium solani and pea endocarp tissue prevent fungal spores from inducing phytoalexin production. Conversely, preinduced host resistance responses are not readily transported from the plant across the membrane barrier to Fusarium macroconidia.Crude enzyme extracts from pea endocarp tissues partially degrade Fusarium solani f. sp. phaseoli cell walls. Activities of the glycosidic enzymes, chitinase, beta-1,3-glucanase, chitosanase, beta-D-N-acetylglucosaminidase, beta-D-N-acetylgalactosaminidase, beta-D-glucosidase, alpha-D-glucosidase, and alpha-D-mannosidase, were detected in pea endocarp tissue. If pods are challenged with Fusarium spores or chitosan, the chitinase activity of the infected tissue remains higher than water-treated pods 0.5 to 6 hours after treatment. The beta-1,3-glucanase activity increases within 6 hours in both inoculated and control tissue. Chitosanase activity was lower in tissue treated with Fusarium solani f. sp. pisi, f. sp. phaseoli or chitosan than in water-treated control tissue. Thus, the pea tissue contains glycosidic enzymes with the potential to degrade the major compounds of the Fusarium cell walls.

Journal Article↗

PubMed1988

Catabolism of Cyanogenic Glycosides by Purified Vicianin Hydrolase from Squirrel's Foot Fern (Davallia Trichomanoides Blume).

Vicianin hydrolase, which catalyzes the hydrolysis of vicianin (K(m), 4.9 millimolar) to (R)-mandelonitrile and vicianose at an optimum pH of 5.5, was extensively purified from the young fronds and fiddleheads of the squirrel's foot fern (Davallia trichomanoides Blume) using DEAE-cellulose and Ultrogel HA chromatography. The native molecular weight of the enzyme was 340,000, and the isoelectric point was 4.6 to 4.7. SDS-PAGE analysis yielded three polypeptides with molecular weights of 56,000, 49,000, and 32,500. The enzyme hydrolyzed only a narrow range of glycosides and, among cyanogenic glycosides, exhibited a strict requirement for (R)-epimers and a preference for disaccharides over monosaccharides. (R)-Amygdalin, (R)-prunasin and p-nitrophenyl-beta-d-glucoside were hydrolyzed at 27, 14, and 3%, respectively, of the rate of vicianin hydrolysis. Mixed substrate studies showed that (R)-vicianin, (R)-prunasin, and p-nitrophenyl-beta-d-glucoside competed for the same active site. The enzyme was significantly inhibited by castanospermine, delta-gluconolactone, and p-chloromercuriphenylsulfonate. Failure to recognize concanavalin A-Sepharose 4B and to stain with periodic acid-Schiff reagent indicated that the enzyme was not a glycoprotein.

Journal Article↗

PubMed1981

Heterogeneity of the glycans O-glycosidically linked to the hinge region of secretory immunoglobulins from human milk.

Pure secretory immunoglobulin A was isolated from human milk by fractionation in gradients of pH and (NH4)2SO4 concentration followed by gel filtration. The hinge region containing all the O-glycosidically linked oligosaccharides was isolated en bloc after trypsin and pepsin hydrolysis and separated by gel filtration. The mixture of O-glycosidically linked oligosaccharides contained N-acetylneuraminic acid (NeuAc), fucose (Fuc), galactose (Gal), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) in the molar ratio of 0.2:0.5:2.5:2:1 respectively. After beta-elimination several oligosaccharides were separated by a combination of ion-exchange chromatography and gel-filtration chromatography. The complete structure of four of these oligosaccharides was determined by methanolysis, methylation and mass spectrometry. The structure of the four oligosaccharides which are linked to serine or threonine residues of the hinge region are as follows: beta-Gal-(1 leads to 3)-GalNAc-ol; alpha-HeuAc-(2 leads to 3)-beta-Gal-(1 leads to 3)-GalNAc-ol; beta-Gal-(1 leads to 3)-[beta-GlcNAc-(1 leads to 6)]-GalNAc-ol; beta-Gal-(1 leads to 3)-[beta-Gal-(1 leads to 4)-beta-GlcNac-(1 leads to 6)]-Gal-NAc-ol. These oligosaccharides are more complex and heterogenous than the oligosaccharides linked to serine residues of the hinge region from myeloma serum immunoglobulin A1.

Amino Acids↗

PubMed1977

Accumulation of radioactive cardiac glycosides by various brain regions in relation to the dysrhythmogenic effect.

Ouabain was administered at a loading dose of 3 mug/kg followed by an infusion at a rate of 1 mug/kg-1 min-1 in order to produce severe dysrhythmia in dogs within 60 minutes. Similarly, digitoxin at a loading dose of 9 mug/kg followed by an infusion at a rate of 3 mug kg-1 min-1 was administered to compare its effect with that of ouabain. 2 During the 60 min experimental period, the plasma concentrations gradually rose with the continuous infusion of these drugs. However, in comparison to the 60 min plasma value of 119+/-20 pmol/ml for ouabain and 177+/-68 pmol/ml for digitoxin, the cerebrospinal fluid (CSF) concentrations for these drugs at this time were less than 5 pmol/ml. 3 Upon termination of the experiment at 60 min it was found that kidney, liver, heart, adrenal, and the non-neural tissue in the brain such as pituitary and choroid plexus concentrated ouabain and digitoxin to give high tissue to plasma ratios. However, various neural areas of the brain (cerebellum, mesencephalon, hypothalamus, pons, and medulla) showed no preferential localization or uptake of these two glycosides. 4 Concentration of ouabain and digitoxin by the choroid plexus does not seem to affect the ionic composition of the CSF. 5 It was concluded that sampling the large areas of neural tissue above could provide no evidence for local accumulation of digitalis glycosides that might account for a central nervous system origin of digitalis-induced cardiac arrhythmias.

Animals↗

PubMed1996

Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco-2) cells.

1. Human intestinal epithelial Caco-2 cells have been used to investigate the transepithelial permeation of the cardiac glycoside, digoxin. 2. Transepithelial basal to apical [3H]-digoxin flux exceeds apical to basal flux, a net secretion of [3H]-digoxin being observed. At 200 microM digoxin, net secretory flux (Jnet) was 10.8 +/- 0.6 nmol cm-2 h-1. Maximal secretory flux (Jmax) of vinblastine was 1.3 +/- 0.1 nmol cm-2 h-1. Cellular uptake of digoxin was different across apical and basal cell boundaries. It was greatest across the basal surface at 1 microM, whereas at 200 microM, apical uptake exceeded basal uptake. 3. Net secretion of [3H]-digoxin was subject to inhibition by digitoxin and bufalin but was not inhibited by ouabain, convallatoxin, and strophanthidin (all 100 microM). Inhibition was due to both a decrease in Jb-a and an increase in Ja-b. Uptake of [3H]-digoxin at the apical surface was increased by digitoxin and bufalin. All cardiac glycosides decreased [3H]-digoxin uptake at the basal cell surface (except for 100 microM digitoxin). 4. The competitive P-glycoprotein inhibitors, verapamil (100 microM), nifedipine (50 microM) and vinblastine (50 microM) all abolished net secretion of [3H]-digoxin due to both a decrease in Jb-a and an increase in Ja-b. Cellular accumulation of [3H]-digoxin was also increased across both the apical and basal cell surfaces. I-Chloro-2,4,-dinitrobenzene (10 microM), a substrate for glutathione-S-transferase and subsequent ATP-dependent glutathione-S-conjugate secretion, failed to inhibit net secretion of [3H]-digoxin. The increase in absorptive permeability Pa-b (= Ja-b/Ca) and cellular [3H]-digoxin uptake upon P-glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP-dependent extrusion at the apical surface. 5. A model for [3H]-digoxin secretion by the intestinal epithelium is likely to involve both diffusional uptake and Na(+)-K+ pump-mediated endocytosis, followed by active extrusion at the apical membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.).

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3'' and 6'' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3'', 4'' or 6''. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3''- and 6''HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6'' followed by acylation at position 3''. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.

Acyltransferases↗

PubMed1972

Binding of the cardiac glycoside ouabain to intact cells.

1. Measurements were made of the binding of [(3)H]ouabain to a variety of cell types.2. Two components of binding could usually be distinguished: a component that saturated at low glycoside concentrations and a component that increased up to the highest ouabain concentrations examined.3. Detailed studies with HeLa cells and kidney slices from guinea-pigs showed that the saturable component is probably associated with inhibition of the Na pump. The main evidence for this is (a) at low concentrations of ouabain there is a close correspondence between the concentration of ouabain giving half-maximum binding and the concentration giving half-maximum inhibition of the Na pump; (b) at low glycoside concentrations, binding precedes inhibition of the Na pump; (c) the rate of binding is very sensitive to external K ions, being highest in the absence of K; (d) binding is reversible and the release of ouabain is associated with reactivation of the Na pump, (e) binding is reduced in the absence of Na ions and in the presence of metabolic inhibitors; (f) binding has a Q(10) of about 4; and (g) in the presence of Na and ATP, lysed HeLa cells bind a similar amount of ouabain and the binding is sensitive to K ions.4. The linear component of binding does not seem to involve the Na pump and it may reflect uptake of ouabain into the cell interior. It has a Q(10) of 2.5 and is unaffected by K concentrations which have a large effect on the saturable component.5. Bound ouabain could be removed from HeLa cells by low pH, trichloroacetic acid, urea, high temperatures and 100% ethanol. These agents did not distinguish between the two components of binding.6. Criteria are developed for estimating the number of Na pumping sites in cells and the data for ouabain-binding to a number of cells is compared with the activity of the (Na + K)-activated ATPase in the same tissues. Although the number of pumping sites varies from less than 1/mu(2) to 1500/mu(2) of membrane, the turnover at these sites seems to be fairly constant between 3,500 and 15,000 min(-1) at 35 degrees C.

Adenosine Triphosphatases↗

PubMed1996

Effects of cardiac glycosides on excitation-contraction coupling in frog skeletal muscle fibres.

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1-2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac-type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha-acetoxy-6-chloro-4, 6-pregnadiene-3,20-dione; 5 microM), a Na(+)-K(+)-ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid-point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.

Animals↗

PubMed1998

Evidence that a New Antibiotic Flavone Glycoside Chemically Defends the Sea Grass Thalassia testudinum against Zoosporic Fungi.

Significantly fewer thraustochytrid protists (zoosporic fungi) were observed in association with healthy leaf tissue of the marine angiosperm Thalassia testudinum than in association with sterilized samples that were returned to the collection site for 48 h. In support of the hypothesis that sea grass secondary metabolites were responsible for these differences, extracts of healthy T. testudinum leaf tissues inhibited the growth of the co-occurring thraustochytrid Schizochytrium aggregatum and deterred the attachment of S. aggregatum motile zoospores to an extract-impregnated substrate. By using S. aggregatum for bioassay-guided chemical fractionation, a new flavone glycoside was isolated and structurally characterized as luteolin 7-O-beta-d-glucopyranosyl-2"-sulfate. Whole-leaf tissue concentrations of this metabolite (4 mg/ml of wet leaf tissue) inhibited S. aggregatum attachment, and a significantly lower concentration (270 mug/ml) reduced thraustochytrid growth by 50%, suggesting that natural concentrations are at least 15 times greater than that needed for significant microbiological effects. These results offer the first complete chemical characterization of a sea grass sulfated flavone glycoside and provide evidence that a secondary metabolite chemically defends T. testudinum against fouling microorganisms.

Journal Article↗

PubMed1997

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA.

We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: 3389-3395, 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the epsilon-rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin.

Anthracyclines↗

PubMed2002

Evidence for temporal regulation of the two Pseudomonas cellulosa xylanases belonging to glycoside hydrolase family 11.

Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or beta-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

Acetates↗

PubMed1986

Urinary excretion of hydroxylysine and its glycosides in Alport's syndrome and several other glomerulopathies.

Alport's syndrome probably is a molecular disorder of basement membrane composition. Investigation of urine on basement membrane components such as hydroxylysine and its glycosides, glucosylgalactosylhydroxylysine and galactosylhydroxylysine, may be helpful for diagnosis of the disease. Urinary specimens of 33 patients and 12 siblings were investigated, and the results were compared with those of 14 healthy adults and of 29 healthy children. The urine of patients with glomerulopathies, occurring during childhood (IgA nephropathy, benign recurrent hematuria, poststreptococcal glomerulonephritis, Henoch-Schönlein nephropathy, membranoproliferative glomerulonephritis, and nephrotic syndrome due to minimal lesions), was also investigated. No marked differences between normal and diseased subjects could be demonstrated, with respect to excretion of hydroxylysine and its glycosides, in contrast to data reported in the literature.

Adolescent↗