Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor

It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing > 5% hexasaccharides (DP6) exhibited > 90% binding, with EC50 values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP6, was dose dependent, with an EC50 value of 5.10 mg/mL (5.15 mu M MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP5 and/or DP6, Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Mechanisms underlying agonist efficacy

Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters (E-max, K-A/EC50, E-max center dot K-A/EC50) determined. The E-max center dot K-A/EC50 parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [S-35]GTP[S] (guanosine 5'-[gamma-[S-35]thio]triphosphate)-binding assay. in this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [S-35]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D-2 dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [S-35]GTP[S] binding follows a pseudo-first-order reaction with [S-35]GTP[S] binding reaching equilibrium after approx. 3 h. The [S-35]GTP[S]-binding event is the rate-deter mining step in the assay. Agonists regulate the maximal level of [S-35]GTP[S] bound, rather than the rate constant for binding. The [S-35]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [S-35]GTP[S] bound rather than the rate of binding.

A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 microM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect on AChE activity but a strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Condensed tannins act against cattle nematodes

The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC50 (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes.

Combinations of parabens at concentrations measured in human breast tissue can increase proliferation of MCF-7 human breast cancer cells

The alkyl esters of p-hydroxybenzoic acid (parabens), which are used as preservatives in consumer products, possess oestrogenic activity and have been measured in human breast tissue. This has raised concerns for a potential involvement in the development of human breast cancer. In this paper, we have investigated the extent to which proliferation of MCF-7 human breast cancer cells can be increased by exposure to the five parabens either alone or in combination at concentrations as recently measured in 160 human breast tissue samples. Determination of no-observed-effect concentrations (NOEC), lowest-observed-effect concentrations (LOEC), EC50 and EC100 values for stimulation of proliferation of MCF-7 cells by five parabens revealed that 43/160 (27%) of the human breast tissue samples contained at least one paraben at a concentration ≥ LOEC and 64/160 (40%) > NOEC. Proliferation of MCF-7 cells could be increased by combining all five parabens at concentrations down to the 50th percentile (median) values measured in the tissues. For the 22 tissue samples taken at the site of ER + PR + primary cancers, 12 contained a sufficient concentration of one or more paraben to stimulate proliferation of MCF-7 cells. This demonstrates that parabens, either alone or in combination, are present in human breast tissue at concentrations sufficient to stimulate the proliferation of MCF-7 cells in vitro, and that functional consequences of the presence of paraben in human breast tissue should be assessed on the basis of all five parabens and not single parabens individually.

Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are influenced by structural features of condensed tannins

Plants containing condensed tannins (CTs) may hold promise as alternatives to synthetic anthelmintic (AH) drugs for controlling gastrointestinal nematodes (GINs). However, the structural features that contribute to the AH activities of CTs remain elusive. This study probed the relationships between CT structures and their AH activities. Eighteen plant resources were selected based on their diverse CT structures. From each plant resource, two CT fractions were isolated and their in vitro AH activities were measured with the Larval Exsheathment Inhibition Assay, which was applied to Haemonchus contortus and Trichostrongylus colubriformis. Calculation of mean EC50 values indicated that H. contortus was more susceptible than T colubriformis to the different fractions and that the F1 fractions were less efficient than the F2 ones, as indicated by the respective mean values for H.contortus F1 = 136.9 ± 74.1 µg/ml; and for H.contortus F2 = 108.1 ± 53.2 µg/ml and for T colubriformis F1 = 233 ± 54.3 µg/ml and F2=166 ± 39.9 µg/ml. The results showed that the AH activity against H. contortus was associated with the monomeric subunits that give rise to prodelphinidins (P < 0.05) and with CT polymer size (P < 0.10). However, for T. colubriformis AH activity was correlated only with prodelphinidins (P < 0.05). These results suggest that CTs have different modes of action against different parasite species.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin > BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

Potent anthelmintic activity of galloylated proanthocyanidins from shea meal (Vitellaria paradoxa)

Proanthocyanindins (PAs) from shea meal (SM), a by-product obtained after lipid extraction of the nuts, contained B-type linkages, had a high ratio of prodelphinidins (73%) and were galloylated (42%). The average polymer size was 8 flavan-3-ol subunits (≈2384 Daltons) and epigallocatechin gallate was the major subunit. Purified PA fractions from SM were tested in vitro for anthelmintic properties against gastrointestinal nematodes from ruminants (H. contortus and T. colubriformis) [1] by the larval exsheathment inhibition assay and from pigs (A. suum) by the larval migration inhibition assay. Results showed that PAs from SM have a potent anthelmintic activity against those parasites similar to white clover (Trifolium repens) flowers (WCF) [1, 2] (EC50 µg/mL; SM: 55.1, 16.5, 75.9; WCF: 37.4, 14.5, 110.1 for A. suum, H. contortus and T. colubriformis respectively). WCF PAs are constituted almost exclusively of prodelphinidin (PD) compared to SM (98% vs. 73%) but do not contained galloylated PAs. Studies [1, 2] have shown that anthelmintic activity of PAs was mainly associated with their PD ratio but our current results suggest that galloylation can be a major factor to anthelmintic activity and SM as a potential nutraceutical anthelmintic feed for controlling parasitic nematodes.

High sodium intake enhances insulin-stimulated glucose uptake in rat epididymal adipose tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity. Methods and Procedures: Male Wistar rats were fed on normal- (0.5% Na+; NS), high- (3.12% Na+; HS), or low-sodium (0.06% Na+; LS) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail-cuff system. An intravenous insulin tolerance test (ivITT) was performed in fasted animals. At the end of each period, rats were killed and blood samples were collected for glucose and insulin determinations. The white adipose tissue (WAT) from abdominal and inguinal subcutaneous (SC) and periepididymal (PE) depots were weighed and processed for adipocyte isolation and measurement of in vitro rates of insulin-stimulated 2-deoxy-d-[H-3]-glucose uptake (2DGU) and conversion of -[U-C-14]-glucose into (CO2)-C-14. Results: After 6 weeks, HS diet significantly increased the BP, SC and PE WAT masses, PE adipocyte size, and plasma insulin concentration. The sodium dietary content did not influence the whole-body insulin sensitivity. A higher half-maximal effective insulin concentration (EC50) from the dose - response curve of 2DGU and an increase in the insulin-stimulated glucose oxidation rate were observed in the isolated PE adipocytes from HS rats. Discussion: The chronic salt overload enhanced the adipocyte insulin sensitivity for glucose uptake and the insulin-induced glucose metabolization, contributing to promote adipocyte hypertrophy and increase the mass of several adipose depots, particularly the PE fat pad.

The role of endothelin pathway in modulation of airway reactivity to methacholine in C57Bl/6 and BALB/c mice

Endothelin peptides have been shown to increase cholinergic neurotransmission in the airway. Genetic differences in airway responsiveness to methacholine where reported in mice. The present study compared the airway reactivity to methacholine in C57Bl/6 and BALB/c mice, the involvement of endothelin on this reactivity and endothelin levels in lung homogenates. Whole airway reactivity was analyzed by means of an isolated lung preparation where lungs were perfused through the trachea with warm gassed Krebs solution at 5 ml/min, and changes in perfusion pressure triggered by methacholine at increasing bolus doses (0.1-100 mu g) were recorded. We found that the maximal airway response to methacholine was much greater in C57Bl/6 than in BALB/c (Emax 34 +/- 2 vs 12 +/- 1 cmH(2)O, respectively). Bosentan (mixed endothelin A/B receptor antagonist; 10 mg/kg, i.p., 30 min before sacrifice) reduced lung responsiveness to methacholine in C57Bl/6 (58% at EC50 level) but had no effect in BALB/c mouse strain. This effect seems to be mediated by the endothelin ETA receptor since it was significantly reduced by the selective endothelin ETA receptor antagonist, BQ 123. Immunoreactive endothelin levels were higher in C57Bl/6 than in BALB/c lungs (43 5 vs 19 +/- 5 pg/g of tissue). In conclusion, airway reactivity to methacholine and lung endothelins content varies markedly between C57Bl/6 and BALB/c strains. Endothelins upregulate lung responsiveness to methacholine only in C57Bl/6, an effect achieved through the endothelin ETA receptor. (C) 2008 Elsevier B.V. All rights reserved.