Lipophilic Aglycone Research Articles

Amphiphilic Heparinoids as Potent Antiviral Agents against SARS-CoV-2.

Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.

Perspectives on Saponins: Food Functionality and Applications.

Saponins are a diverse group of naturally occurring plant secondary metabolites present in a wide range of foods ranging from grains, pulses, and green leaves to sea creatures. They consist of a hydrophilic sugar moiety linked to a lipophilic aglycone, resulting in an amphiphilic nature and unique functional properties. Their amphiphilic structures enable saponins to exhibit surface-active properties, resulting in stable foams and complexes with various molecules. In the context of food applications, saponins are utilized as natural emulsifiers, foaming agents, and stabilizers. They contribute to texture and stability in food products and have potential health benefits, including cholesterol-lowering and anticancer effects. Saponins possess additional bioactivities that make them valuable in the pharmaceutical industry as anti-inflammatory, antimicrobial, antiviral, and antiparasitic agents to name a few. Saponins can demonstrate cytotoxic activity against cancer cell lines and can also act as adjuvants, enhancing the immune response to vaccines. Their ability to form stable complexes with drugs further expands their potential in drug delivery systems. However, challenges such as bitterness, cytotoxicity, and instability under certain conditions need to be addressed for effective utilization of saponins in foods and related applications. In this paper, we have reviewed the chemistry, functionality, and application aspects of saponins from various plant sources, and have summarized the regulatory aspects of the food-based application of quillaja saponins. Further research to explore the full potential of saponins in improving food quality and human health has been suggested. It is expected that this article will be a useful resource for researchers in food, feed, pharmaceuticals, and material science.

2-C-Branched mannosides as a novel family of FimH antagonists—Synthesis and biological evaluation

Summary Urinary tract infections (UTIs), which are among the most prevalent bacterial infections worldwide, are mainly attributed to uropathogenic Escherichia coli (UPEC). Because of frequent antibiotic treatment, antimicrobial resistance constitutes an increasing therapeutic problem. Antagonists of the mannose-specific bacterial lectin FimH, a key protein mediating the adhesion of UPEC to human bladder cells, would offer an alternative anti-adhesive treatment strategy. In general, FimH antagonists consist of a mannose moiety and a wide range of lipophilic aglycones. Modifications of the mannose core led to a distinct drop in affinity. A visual inspection of the crystal structure of FimH revealed a previously unexplored cavity surrounded by Ile13, Phe142 and Asp140, which could be reached by functional groups in the equatorial 2-position of the mannose. Here, we describe the synthesis of 2-C-branched mannosides and evaluation of their pharmacodynamic properties. ITC experiments with the selected antagonists revealed a drastic enthalpy loss for all 2-C-branched antagonists, which, however, is partially compensated by an entropy gain. This supports the hypothesis that the target cavity is too small to accommodate 2-C-substituents.

A Strategy for the Improvement of the Bioavailability and Antiosteoporosis Activity of BCS IV Flavonoid Glycosides through the Formulation of Their Lipophilic Aglycone into Nanocrystals

Epimedium-derived flavonoid glycosides are widely used for the prevention of osteoporosis, but these compounds generally exhibit poor membrane permeability and oral absorption. To address these limitations, the bioactive lipophilic aglycone icaritin (ICT) was selected and successfully developed into nanocrystals (ICTN) through the antisolvent-precipitation method. After the parameters in the preparation of ICTN were optimized, the morphology, crystallinity, adsorption of the stabilizers on the ICT surface, and the dissolution of the resulting nanocrystals were characterized. The pharmacokinetics in rat and the in vitro antiosteoporosis activity of serum withdrawn after the oral administration of ICTN to rats on mouse osteoblastic cells were evaluated. Consistent with its good performance in stabilizing the ICT nanosuspension, atomic force microscopy showed that hydroxypropyl methylcellulose (HPMC) exhibits better adsorption on the ICT surface compared with other stabilizers. Needle-shaped crystals (∼ 220 nm in diameter) with a high drug loading (∼ 90%) were generated when 0.16 mL of the ICT acetone solution (10 mg/mL) was injected quickly into 2 mL of the HPMC solution (0.02%, w/w) under ultrasonication for 10 s at room temperature. The thermal analysis demonstrated that the majority of the particles are in their crystalline forms, similarly to the unformulated ICT. After oral administration, ICTN exhibited a faster dissolution rate and significantly faster absorption, as supported by the increased AUC0-36h and Cmax and the reduced Tmax of these nanocrystals compared with the raw suspension (p < 0.05). Compared with blank serum, enhanced proliferation and differentiation activities were observed when serum withdrawn after the oral administration of ICTN in rat was incubated with osteoblast MC3T3-E1 cells. The present delivery system could provide a new promising strategy for BCS IV glycoside of flavonoids or other natural products by formulation of their bioactive lipophilic aglycone forms to enhance oral absorption and in vivo bioactivity.

Effect of C-ring modifications on the cytotoxicity of spirostan saponins and related glycosides

Twelve C-ring modified spirostanyl glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). With the aim of assessing the influence of the hydrophobic character, the conformational flexibility and the stereochemistry of the C-ring functionalities on the cytotoxic activity, a variety of spirostanic aglycones incorporating methylene, methoxyl, α,β-unsaturated ketone and lactone groups were subjected to a linear glycosylation strategy leading to glycosides derived from the 3,6-dipivaloylated β-d-glucoside and the β-chacotrioside moieties. The 3,6-dipivaloylated spirostanyl β-d-glucosides showed moderate to good cytotoxic activity against HL-60, but no significant cytotoxicity against benign blood cells. However, the cytotoxicity of spirostanyl β-chacotriosides was highly dependent on the nature of the C-ring functional groups of the steroidal aglycones. Actually, the chacotrioside-based saponins either with no functionality or bearing a hydrophobic methylene group at C-12 were the most cytotoxic ones against both HL-60 and benign blood cells. On the other hand, the incorporation of very polar functionalities and the opening of the ring C with the consequent loss of rigidity led to a significant drop in the cytotoxicity against HL-60. These results confirm that spirostanyl β-chacotriosides including very lipophilic aglycones are the most cytotoxic ones among their congeners.

Metabonomic study on the cumulative cardiotoxicity of a pirarubicin liposome powder

Pirarubicin (THP) is an anthracycline frequently used in the chemotherapy against acute leukemia, malignant lymphoma and several solid tumors. However, its clinical use is severely limited by the development of a progressive dose-dependent cardiomyopathy that results in irreversible congestive heart failure. To provide a strategy for constraining or minimizing the cumulative cardiotoxicity of THP, a pirarubicin liposome powder (L-THP) was appropriately prepared, and the cumulative cardiotoxicity of L-THP and free THP (F-THP) were investigated on Sprague-Dawley rats after 3 successive doses. Urinary samples for metabonomic study, serum samples for biochemical assay, and heart samples for histopathology test were collected. As a result, the metabonomics-based findings such as PLS-DA plotting showed minimal metabolic alterations in L-THP as compared to F-THP, and correlated with the changes of serum biochemical assay and cardiac histopathology as measurements of damage to heart tissue. Our results confirm that when encapsulated into liposomes, the cumulative cardiotoxicity of THP can be greatly ameliorated. Lipophilic aglycone metabolites of THP associated with redox cycling are cardiotoxic for the possibility of reactive oxygen species (ROS) formation. Also, metabonomic analysis shows that the successive doses of THP will lead to severe metabolic pathways disturbances in the cell energy production. Further, the preliminary efficacy study of L-THP on lung cancer was evaluated in the approach of in vitro cytotoxicity on A549 cells by high content screening (HCS) analysis, and L-THP was found to exhibit better therapeutic index against lung cancer than THP.

Evolution of sodium glucose co-transporter 2 inhibitors as anti-diabetic agents

Background: A critical factor for maintenance of glucose balance is the renal recovery of glucose from the glomerular filtrate mediated primarily by sodium glucose co-transporter 2 (SGLT2). This capacity can be modulated by SGLT2 inhibitors thereby providing a unique insulin independent method of treatment of diabetes. Objective/method: A discussion of the evolution of SGLT inhibitors as inferred from patents published from 2005 to 2009 is prefaced by a brief review of the role of SGLT in glucose transport and the clinical findings illustrating the therapeutic potential of SGLT inhibitors as anti-diabetic agents. These compounds comprise O, C and N-glycosides generated by attachment of an appropriate lipophilic aglycone component to a suitable glucose analogue. Conclusion: The realization that the in vivo potency of O-glucosides was markedly less than that of C-glucosides necessitated a shift in medicinal chemistry focus of the pharmaceutical companies pursuing SGLT2 inhibitors. Particular emphasis is placed on the strategy that each used to circumvent the constraints imposed by prior art while utilizing a common pharmacophore. The role of SGLT2 inhibitors for treatment of diabetes will be established by the outcome of the five compounds in advanced clinical trials.

Monitoring by HPLC of Chamomile Flavonoids Exposed to Rat Liver Microsomal Metabolism

Three major flavonoid chamomile components (quercetin, apigenin-7-O-glucoside and rutin) were subjected to oxidative metabolism by cytochrome P-450 of rat liver microsomal preparations. Changes over time in their respective concentrations were followed using reversed-phase HPLC with UV detection. No clean-up had to be applied as only the specific flavonoid had to be separated from the background components originating from the rat liver microsome.Neither the concentration of apigenin-7-O-glucoside nor that of the diglycoside rutin decreased during one hour of exposure to rat microsomal treatment. In contrast, the concentration of quercetin, a lipophilic aglycon, decreased.Our analytical HPLC results complement the in silico calculated lipophilicity (logP) of these compounds; the relatively high lipophilicity of quercetin appears to predispose it to oxidative metabolism in order to decrease its fat solubility. In contrast the much less lipophilic compounds apigenin-7-O-glucoside and rutin were resistant in vitro to microsomal treatment.

REGULATORY EFFECTS OF MYOENDOTHELIAL GAP JUNCTION ON VASCULAR REACTIVITY AFTER HEMORRHAGIC SHOCK IN RATS

Myoendothelial gap junction (MEGJ), one kind of gap junction between vascular endothelial cell and vascular smooth muscle cell, can transmit electrical and chemical signals to keep the electric and machinery activity synchronism of vasculature. After severe trauma or shock, vascular reactivity to vasoconstrictors or vasodilators is greatly reduced. However, whether MEGJ participates in the regulation of vascular reactivity after hemorrhagic shock, what type of MEGJ is involved, and what is the possible mechanism are unknown. With the hemorrhagic shock Sprague-Dawley rats and their superior mesenteric arteries (SMAs), the effects of 18alpha-glycyrrhetic acid, a lipophilic aglycone that disrupts gap junction plaques, on vascular contractile response to norepinephrine (endothelium-independent vascular constrictor), myricetin (endothelium-dependent vasoconstrictor) and relaxation reactivity to sodium nitroprusside (endothelium-independent vasodilator), and acetylcholine (Ach; endothelium-dependent vasodilator) were observed. Meanwhile, the relationship of the mRNA/protein expression of connexins 37, 40, and 43(Cx40 and Cx43) to the changes of vascular reactivity after hemorrhagic shock and the effect of antisense oligodeoxynucleotide of Cx40 or Cx43 on vascular calcium sensitivity and vascular reactivity were investigated. The results indicated that 18alpha-glycyrrhetic acid antagonized myricetin and Ach-induced SMA reactivity, but had no effect on norepinephrine- and sodium nitroprusside-induced vascular response. The mRNA and protein expression of Cx37 and Cx40 of SMA were negatively associated with the vascular reactivity, whereas Cx43 seemed to be a positive relationship to vascular reactivity. Antisense oligodeoxynucleotide of Cx40 significantly increased the calcium sensitivity, myricetin-induced vasoconstriction, and Ach-induced vasodilation, whereas antisense oligodeoxynucleotide of Cx43 depressed them. It was suggested that MEGJ plays an important role in the regulation of endothelium-dependent vascular reactivity after hemorrhagic shock. The involved types were mainly Cx40 and Cx43. The possible mechanism that Cx40/Cx43 regulates the endothelium-dependent vasoconstrictor reactivity may be related to their regulating effects on the calcium sensitivity of vascular smooth muscle cell.

Experimental Determination of Octanol−Water Partition Coefficients of Quercetin and Related Flavonoids

Octanol-water partition coefficient (log P) values were determined for flavonoids from the flavone, flavonol, flavanone, and isoflavonoid subclasses. Each flavonoid was dissolved in an octanol-water system and allowed to equilibrate, and then both fractions were analyzed by high-performance liquid chromatography. log P was calculated as log[ratio of the concentration in the octanol phase to the concentration in the aqueous phase at pH 7.4]. The aglycons were more lipophilic than any conjugate. The conjugate moiety had a more significant effect on log P than the aglycon moiety. Quercetin was the least lipophilic aglycon (log P = 1.82 +/- 0.32) and, together with kaempferol (log P = 3.11 +/-0.54), gave the most variable results. The isoflavones genistein and daidzein and the isoflavone metabolite equol gave relatively high log P values (3.04 +/- 0.02, 2.51 +/- 0.06, and 3.20 +/- 0.13, respectively), while glycitein had an unexpectedly low value of 1.97 +/- 0.05. The conjugation characteristics and hydroxylation pattern were the most important determinants of log P in general, and log P was highly variable within the flavonoid subclass. The results are discussed in terms of further understanding of the in vivo fate of the flavonoids as important dietary bioactives.

Bioavailability of Quercetin in Pigs Is Influenced by the Dietary Fat Content

The flavonol quercetin is one of the most prevalent flavonoids found in edible plants. In this study, the influence of dietary fat on oral bioavailability of quercetin was investigated. Quercetin (30 micromol/kg body weight) was administered either as the lipophilic aglycone or as the more hydrophilic quercetin-3-O-glucoside in test meals differing in fat content (3, 17, or 32 g fat/100 g diet) to growing pigs. Blood samples were drawn repeatedly over a 24-h period and analyzed by HPLC. The main metabolite found in plasma was always conjugated quercetin. Quercetin bioavailability from each diet was always higher from the glucoside than from the aglycone. Irrespective of the chemical form applied, the bioavailability of quercetin was higher in the 17% fat diet compared with the 3% fat diet (P < 0.05). No further effect on bioavailability was observed when the flavonols were administered with diets containing 32% fat. The elimination of quercetin was significantly delayed after its application with fat-enriched diets (P < 0.05). Thus, in addition to the chemical form of the flavonol, the fat content of the diet influences oral bioavailability of quercetin.

Human UDP-glucuronosyltransferase 2B7.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins, localized in endoplasmic reticulum (ER) and nuclear membranes, which catalyze the confugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GlcUA) as the sugar donor. The major function of glucuronidation is to change hydrophobic compounds into hydrophilic derivatives, a process which facilitates their detoxification and excretion. However, it is also widely recognized that glucuronidation can result in compounds which are biologically active or demonstrate increased toxicity. UGTs, like other drug-metabolizing enzymes, have been postulated to be involved in controlling the steady state concentrations of nuclear receptor ligands for interactions with nuclear receptors [1,2]. One of the isoforms from the UGT2B subfamily, UGT2B7, has been found to be a major human UGT2B isoform, involved in the glucuronidation of a variety of endogenous compounds and xenobiotics. In this review, we included all available information from our studies and those of other investigators on a) the history of the identification and expression of UGT2B7 in human tissues, b) the substrate specificity of UGT2B7, c) the extrahepatic localization of UGT2B7 d) the nuclear localization of UGT2B7 and e) characterization of the UGT2B7 gene and promoter.

Inhibition of leukocyte eicosanoid generation and radical scavenging activity by gnaphalin, a lipophilic flavonol isolated from Helichrysum picardii.

The lipophilic aglycone 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphalin) isolated from the aerial flowering parts of Helichrysum picardii Boiss. & Reuter (Asteraceae) was tested for interactions with the cyclo-oxygenase and 5-lipoxygenase pathways of arachidonate metabolism in stimulated rat peritoneal leukocytes, and for its effects on leukocyte granular enzyme release, cell viability and interactions with reactive oxygen species. Gnaphalin dose-dependently inhibited generation of the cyclo-oxygenase metabolite thromboxane B2 (IC50 = 39.9 +/- 3.9 microM), and of the 5-lipoxygenase metabolite leukotriene B4, although the potency was two-fold less (IC50 = 81.8 +/- 12.9 microM). At concentrations of 6 to 320 microM, gnaphalin did not affect secretion of the pro-inflammatory enzymes lysozyme, myeloperoxidase and beta-glucuronidase from the neutrophil secretory granules, and did not scavenge hydrogen peroxide or hypochlorous acid. However, gnaphalin effectively scavenged superoxide radicals generated in the hypoxanthine/xanthine oxidase system (IC50 = 40 microM) and by PMA-stimulated leukocytes (> 60% at 500 microM), directly inhibited xanthine oxidase (85% at 395 microM) and inhibited Fe(3+)-ascorbate-induced liposomal peroxidation (IC50 = 215 microM). Thus, like some other flavonoids found in medicinal herbs, gnaphalin possesses an array of potentially beneficial anti-eicosanoid and free-radical scavenging properties which may alongside other constituents contribute to the claimed medicinal properties of the plant from which it is derived.

STRUCTURAL AND FUNCTIONAL STUDIES OF UDP-GLUCURONOSYLTRANSFERASES*

UDP–Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body.In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER.Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP–GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain.The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

Analyses of the Molecular Mechanism of Adriamycin-Induced Cardiotoxicity

The molecular basis of the adriamycin(AQ)-dependent development of cardiotoxicity is still far from being clear. In contrast to our incomplete understanding of the organ-specific mechanism mitochondria are unequivocally accepted as the locus where the molecular disorder is triggered. A growing number of reports intimate the establishment of unbalanced oxygen activation through heart mitochondria in the presence of anthraquinones. In fact, in contrast to liver mitochondria, isolated heart mitochondria have been unequivocally shown to shuttle single electrons to AQ, giving rise to O 2 ̇− formation by autoxidizing AQ ̇ semiquinones. Earlier we have demonstrated the involvement of the exogenous NADH dehydrogenase in this deleterious electron deviation from the respiratory chain. This enzyme that is associated with complex I of the respiratory chain catalyzes the oxidation of cytosolic NADH. AQ activation through isolated heart mitochondria was reported to require the external addition of NADH, suggesting a flux of reducing equivalents from NADH to AQ in the cytosol. Unlike heart mitochondria, intact liver mitochondria, which are lacking this NADH-related pathway of reducing equivalents from the cytosol to the respiratory chain, cannot be made to activate AQ to semiquinones by NADH or any other substrate of respiration. It appears, therefore, that the exogenous NADH dehydrogenase of heart mitochondria exerts a key function in the myocardial toxicogenesis of anthraquinones via oxygen activation through semireduced AQ. Assessing the toxicological significance of the exogenous NADH dehydrogenase in AQ-related heart injury requires analysis of reaction products and their impact on vital bioenergetic functions, such as energy gain from the oxidation of respiratory substrates. We have applied ESR technique to analyze the identity and possible interactions of radical species emerging from NADH-respiring heart mitochondria in the presence of AQ. The following metabolic steps occur causing depression of energy metabolism in the cardiac tissue. After one-electron transfer to the parent hydrophilic anthraquinone molecule destabilization of the radical formed causes cleavage of the sugar residue. Accumulation of the lipophilic aglycone metabolite in the inner mitochondrial membrane diverts electrons from the regular pathway to electron acceptors out of sequence such as H 2O 2. HO ̇ radicals are formed and affect the functional integrity of energy-linked respiration. The key and possibly initiating role of the exogenous NADH dehydrogenase of cardiac mitochondria in this reaction pathway provides a rationale to explain the selective cardiotoxic potency of the cytostatic anthraquinone glycosides.

Effect of fluorescence intensifiers on the fluorodensitometric determination of flavones and flavonols after detection with diphenylboric acid 2-aminoethyl ester

The measurement of the fluorescence emitted by flavones and flavonols in situ on thin-layer chromatography plates can be improved by dipping the developed plates in appropriate solutions. The effect of different substances on the intensity and stability of the fluorescence of 14 flavone and 26 flavonol derivatives detected with diphenylboric acid 2-aminoethyl ester (Naturstoffreagens A, NA) is described. Silicone oil or paraffin is most appropriate for lipophilic flavonoids while the more hydrophilic polyethylene glycol 4000 is most appropriate for less lipophilic aglycones as well as glycosides. Authentic reference standards in appropriate concentrations must be measured on the same plate as the experimental sample since flavonoids demonstrate specific fluorescence behaviour and because in situ fluorodensitometry measurements are difficult to reproduce. The correlation between fluorescence and molecular structure is discussed briefly. The test conditions established are an improvement on the accepted in situ fluorodensitometry determination of flavonoids.

Spin-labelled glycolipid analogues: d-glucose series

Neoglycolipids bearing a paramagnetic probe in their lipophilic aglycon have been prepared. All belong to the d-glucose series, both anomers for the glucoside representatives, respectively β and α anomers in the S- and C-glucosyl series. Two different types of radical sites have been used, a relatively short-lived imino N-oxyl group for glucosides and a more stable N-acylamino N-oxyl moiety in the other cases. EPR spectra of these radical species afforded information on the conformation of the lipophilic chain in the vicinity of the paramagnetic probe.

Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and sap-C. Activator proteins stimulate lactosylceramide hydrolysis.

Two exo-beta-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-beta-galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-beta-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2 > C4 > C6 > C8 > C10 > C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-beta-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-beta-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561-572]. GM1-beta-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.

Specificity of human glucosylceramide beta-glucosidase towards synthetic glucosylsphingolipids inserted into liposomes. Kinetic studies in a detergent-free assay system.

The behaviour of highly purified glucosylceramide beta-glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560-3563] was investigated in the absence of detergents with structurally modified glucosylceramides inserted into unilamellar liposomes. The reaction between the water-soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl-N-acetyl-sphingosines (D-erythro and L-threo) were better substrates than the corresponding glucosylceramides. The L-threo derivatives were poorer substrates with higher apparent Km values than the corresponding D-erythro derivatives. For glucosyl-3-keto-ceramide and glucosyl-dihydro-ceramide (D-erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl-N-acetyl-sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. D-erythro-Ceramide and D-galactosyl-N-acetyl-D-erythro-sphingosine inhibited the hydrolysis of amphiphilic glucosylceramide but not that of water-soluble 4-methyl-umbelliferyl-beta-glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane-bound substrate. Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins. Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30-40-fold) of the glucosylceramide beta-glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3-10-fold).

Exkret-Flavonoide bei H�heren Pflanzen arider Gebiete

Flavonoids generally occur in higher plants as water-soluble glycosides. However, lipophilic flavonoid aglycones can be excreted by glandular trichomes or extruded through the cuticle. They are accumulated on the plant surface as constituents of leaf resins or thin epicuticular layers. External flavonoid aglycones are found in various families throughout the higher plants, but appear to be most abundant in theAsteraceae. They occur relatively frequently in plants of (semi-)arid habitats. It is advantageous for chemotaxonomic studies that the structural diversity of exudate flavonoids is normally greater than that of the tissue glycosides in these plants.