Partial characterization of neurotoxic esterase of human placenta

Neurotoxic esterase (NTE), the putative target for organophosphorus-induced delayed axonopathy, has been found in preparations of human placenta. The activity was primarily found in membrane-enriched fractions rather than high-speed supernatant. NTE was solubilized from a mixture of mitochondrial and microsomal membranes with Triton X-100. The crude and solubilized activities had inhibitor characteristics similar to preparations from avian brain. Because of the similarities to NTE from brain and ready availability, human placenta may be an ideal source for the bulk purification of a human form of the enzyme.     

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.     

Chromatographic characterization of neurotoxic esterase

Neurotoxic esterase (neuropathy target enzyme, NTE) is an enzyme whose irreversible inhibition is the apparent first step in the induction of organophosphorus-induced delayed neuropathy. NTE is an integral membrane protein and thus must be solubilized before isolation can be attempted. This study describes solubilization of active chicken brain NTE with the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and characterization of the detergent-solubilized enzyme by gel exclusion chromatography. When detergent-solubilized membranes were chromatographed on Sepharose gel exclusion media, NTE activity eluted with an apparent molecular weight of 880-970 kD. When [3H]diisopropylphosphorofluoridate-radiolabeled membranes and unlabeled microsomal membranes were CHAPS-solubilized, combined and chromatographed on Sepharose 4B, NTE activity coeluted with two radiolabeled proteins (Mr = 148 kD and Mr = 112 kD using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with reducing conditions). Another radiolabeled protein (Mr = 92 kD) coeluted exclusively with inhibitor-resistant esterase activity. This study provides strong evidence that the 148 and 112 kD proteins are subunits of a multicomponent NTE complex.     

The effect of detergents on the purified flavin-containing monooxygenase of mouse liver, kidney and lungs.

1. The effect of various commonly used membrane solubilizing detergents on the activity of the microsomal xenobiotic metabolizing enzyme, the flavin-containing monooxygenase (FMO) purified from mouse liver, kidney and lungs was determined. 2. Regardless of the type of detergent used, the effect on the enzyme activity was variable depending on the type of substrate used. 3. Emulgen 911 concentrations of up to 10% had very little effect on thiobenzamide-S-oxidation by liver, kidney or lung FMO. 4. While Emulgen 911 increased substrate dependent NADPH oxidation rate by thiourea and thioacetamide, it drastically reduced the activity toward the organophosphorous compounds, disulfoton, fenthion, fonofos and phorate at low concentrations. 5. Activities of fenthion, phorate and fonofos were decreased by 80, 65 and 55% by the inclusion of 0.25% Emulgen 911 in the assay mixture. 6. This decline in FMO activity for phorate was evident regardless of the type of detergent used. In contrast, thiourea dependent NADPH oxidation rate in the presence of various detergents was variable. 7. Thiourea oxidation rate was decreased by cholate and Zwittergent 3-12, whereas it was increased in the presence of Emulgen 911, Triton X-100 and Tween 20. 8. This study shows that before FMO activity is determined in the presence of detergents their effects should be carefully evaluated.     

Modulation of neurotoxic esterase activity in vitro by phospholipids

Neurotoxic esterase (NTE), the proposed molecular site for the initiation of organophosphorus-induced delayed neuropathy, is a membrane-associated enzyme. NTE activity was solubilized from chicken brain microsomal membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate and partially separated from other solubilized hen brain esterases by DEAE-Sephacel anion-exchange chromatography using stepwise increases in salt concentration; however, there was poor recovery of NTE activity and only a slight increase in NTE specific activity. NTE activity in the "high salt" fraction (i.e., the NTE-enriched fraction) was markedly activated by a heat-stable factor(s) present in other fractions eluted from the column. This activating factor was extracted with organic solvents, suggesting that it may be lipid. In a related study, purified phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were also found to activate the partially separated NTE activity in a concentration-dependent manner while phosphatidyl-inositol was found to inhibit the same partially separated NTE fraction in a concentration-dependent manner. The results suggest that lipids may modulate NTE activity and that the loss of lipid cofactors during chromatographic separations may underlie some of the difficulties encountered in isolation of active NTE.     

UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside. Differences in activation and induction properties in rat and mouse liver

Glucuronidation of digitoxigenin-monodigitoxoside (DT1), a metabolite of the cardiac glycoside digitoxin, is mediated by the microsomal isozymes, UDP-glucuronosyltransferase(s) (UDP-GT). The present studies examined the activation and induction properties of UDP-GT activity toward DT1 in hepatic microsomes of rats and mice. When compared to enzyme activity present in native (latent) microsomes of the rat (0.104 +/- 0.010 nmol/min/mg of protein), the activity toward digitoxigenin-monodigitoxoside in mouse native microsomes was 3.5-fold higher (0.379 + 0.44 mumol/min/mg of protein). After treatment with ionic (sodium cholate), zwitterionic [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)], or nonionic (Emulgen 911, Triton X-100) detergents, or with UDP-N-acetylglucosamine, enzyme activity in rat microsomes remained unchanged. In contrast, UDP-GT activity (DT1) in mouse liver microsomes treated with detergents or with the nucleotide was increased 2-3-fold above native enzyme activity. Pretreatment of rats with the microsomal enzyme inducers, 3-methylcholanthrene and phenobarbital, had no effect on this enzyme activity, whereas pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone (DEX) increased enzyme activity toward DT1 800 and 380%, respectively. These findings support the hypothesis that PCN and DEX induce a unique form of UDP-GT in the rat that selectively glucuronidates DT1. In marked contrast, the activity of this enzyme in mouse liver was not affected by pretreatment with any of the microsomal inducers, including PCN and DEX. In both rat and mouse, the P-450p-dependent N-ethylmorphine demethylase activity was increased 10-15-fold in PCN-pretreated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Separation of paraoxon and mipafox sensitive esterases by sucrose density gradient sedimentation

Paraoxon- and mipafox-sensitive phenyl valerate (PV) hydrolases found in chicken nervous tissue were solubilized by Triton X-100 and separated by sucrose density gradient centrifugation. Enzyme activity from chick embryo brain with properties of neurotoxic esterase (NTE, insensitive to 40 microM paraoxon, sensitive to 50 microM mipafox) migrated in a 9S peak.     

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).     

Optimal detergent activation of rat liver microsomal UDP-glucuronosyl transferases toward morphine and 1-naphthol: contribution to induction and latency studies.

The detergent-activation profiles of UDP-glucuronosyl transferases (UGTs, EC 2.4.1.17) toward 1-naphthol and toward morphine have been determined: three non-ionic detergents, Triton X-100, Brij 58 and Lubrol Px and one zwitterion detergent, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonic acid (CHAPS) were studied. The results showed that marked inhibition of 1-naphthol-UGT and morphine UGT activities occurred with high concentrations of Triton X-100. Lubrol Px, at high concentrations, inhibited 1-naphthol-UGT but not morphine-UGT. It appeared that the detergent/protein ratio suitable for optimal activation of both isoenzymes was limited to 0.2 for these detergents. In contrast, Brij 58 and CHAPS displayed optimal activation of the two enzymes for a large range of detergent/microsomal protein ratios (respectively from 0.2 to 1 and from 0.4 to 1), making them the most suitable for induction and/or latency studies of both isoenzymes. The influence of maximal activation status on the effect of 3-methylcholanthrene and phenobarbital treatment on morphine-UGT and 1-naphthol-UGT activity has also been evaluated. The findings provided evidence that detergent-activation profiles and optimal detergent-activated versus "native" UGT activity determination give crucial informations about the characteristics of a given isoenzymic form of UGT, i.e. its sensitivity to specific alterations of the phospholipid environment, its latency and its inducibility.     

Kinetics of heat inactivation of phenyl valerate hydrolases from hen and rat brain

Heat inactivation was studied at 45, 50, 55, and 60 degrees for all of the phenyl valerate hydrolases (PVase), including neurotoxic esterase (NTE) and inhibitor-resistant esterase (IRE), in homogenates of hen or rat brain or in preparations of hen brain microsomal membranes. Hen and rat brain homogenates were prepared in buffer (50 mM Tris/0.20 mM EDTA, pH 8.00, at 25 degrees). Hen brain microsomes were suspended either in buffer or in aqueous dimethyl sulfoxide (DMSO, 40%, w/v), or solubilized either in aqueous Triton X-100 (0.10%, w/v) or in 40% (w/v) DMSO. Enzyme activities were measured at 37 degrees using phenyl valerate as substrate. Each enzyme activity in all of the preparations exhibited biphasic heat inactivation kinetics. Apparent rate constants were calculated for the fast (kf) and slow (ks) reactions, along with the relative amounts of activity in each component (Af, As) expressed as percentages of the total activity. For a given preparation and temperature, respective values of kf or ks were similar for PVase, NTE, and IRE, with a mean kf/ks ratio of 52 across all preparations. Af and As were a function of temperature. Mean values of the apparent activation energies (Ea) for all activities and preparations were 44 and 25 kcal/mol for the fast and slow inactivation reactions respectively. These results indicate that all phenyl valerate hydrolases in hen and rat brain undergo a common heat-induced structural change leading to loss of enzymic activity.     

Effects of nondenaturating zwitterionic detergent CHAPS, 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulfonate, on rat liver mitochondrial and microsomal monoamine oxidase

It has been reported that monoamine oxidase (MAO) activity (EC1.4.3.4) and, in general, enzymes possessing cationic substrates, were activated and inhibited by anionic and cationic detergents, respectively. In order to examine this hypothesis, the effect of the zwitterionic detergent CHAPS 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulphonate was studied in comparison with the effects of cationic, anionic, and non-ionic detergents. The non-denaturating zwitterionic detergent CHAPS was used to solubilise rat liver monoamine oxidase MAO (EC1.4.3.4) of mitochondrial and microsomal origin; the solubilisation conditions, purification, inhibition and kinetic studies were then determined. These results are compared with those previously obtained with the non-ionic detergent Triton X-100, which would also be expected to have no net charge, and are interpreted in terms of specific ionic effects.     

Reconstitution of alpha 1-adrenoceptors having high affinity for prazosin.

Using a variety of detergents we find that soluble alpha 1-adrenoceptor recovery from aortic and hepatic membranes is markedly enhanced if the receptor is first prelabelled with prazosin. Moreover, prelabelling prevents the reduction in prazosin affinity induced by solubilizing concentrations of digitonin, possibly by stabilizing the receptor's conformation. 20-25% of alpha 1-adrenoceptors solubilized in sodium cholate were reconstituted into brain lipids. Specific [3H]prazosin binding to the reconstituted receptor was saturable and of high affinity (KD = 0.019 +/- 0.008 nM). We conclude that prelabelling is essential to preserve the receptor in detergents. However, once the detergent is removed brain lipids alone are able to maintain the receptor in a form with high affinity for prazosin.     

Interactions of rat brain acetylcholinesterase with the detergent Triton X-100 and the organophosphate paraoxon.

Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci.41, 8-20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the (app)V(max) for hydrolysis of acetylthiocholine, without affecting the (app)K(m) or the (app)K(ss). However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM-100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the (app)k(i). These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels.     

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Muscarinic receptors from rat forebrain have been solubilized by Lubrol PX, lysophosphatidylcholine (LPC), digitonin and cholate/1 M sodium chloride. The overall level of solubilization was characterized using receptors prelabelled with an irreversible antagonist. The recovery of nondenatured soluble binding activity was estimated using reversible tritiated antagonists. All these detergents solubilized 60-85% of the total binding sites. In Lubrol PX most of the receptors were recovered in a denatured form. In the other detergents 30-90% of the solubilized receptors were stable and capable of binding reversible [3H]-antagonists with high affinity. The hydrodynamic properties of the soluble receptors have been examined by gel filtration and sucrose gradient centrifugation in H2O and D2O. The soluble receptors in Lubrol PX, lysophosphatidylcholine and cholate were, in general, heterogeneous as regards their molecular size. Estimates of the molecular weight after correction for bound detergent, varied from 82,000 to 134,000. Conditions were identified under which the receptor was largely monodisperse, and the estimates of molecular weight agreed with values (ca. 83,000) from sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The amount of bound detergent could not be calculated for the digitonin-muscarinic receptor complex which had an estimated overall median molecular weight of about 290,000. It is concluded that a subpopulation of muscarinic receptors from the rat forebrain is capable of existing in a monomeric soluble form and binding ligands. There is also evidence that complexes with other proteins can exist, but their specificity and functional relevance are not known.     

微粒体谷胱甘肽转移酶的纯化

本文采用N—乙基顺丁烯二酰亚胺活化、Triton X—100增溶、羟基磷灰石和离子交换柱层析从Wistar大鼠肝脏中提纯了微粒体谷胱甘肽转移酶。SDS—聚丙烯酰胺凝胶电泳为一条带。氨基末端为丙氨酸。由不同角度鉴定酶制备物的纯度,认为它达到了均一,提纯倍数为28,回收率38％,比活性为15μmol·min~(-1)·mg~(-1),符合进一步进行性质研究的要求。它的最适pH为6.8,最适温度为30℃。     

Electrophoretic properties of mitochondrial monoamine oxidase in monkey liver

Monkey liver monoamine oxidase (MAO) was predominantly the B-form enzyme from the observed differences in substrate specificities and differences in sensitivities to MAO inhibitors. It is known that a MAO inhibitor, pargyline, binds to MAO irreversibly in the molar ratio of 1:1. 3H-pargyline was used as a marker to determine the existence of MAO. The molecular weight of MAO in monkey liver mitochondria was investigated by SDS-polyacrylamide gel electrophoresis after solubilization of 3H-pargyline binding mitochondria with 6% sodium dodecyl sulphate (SDS). The subunit molecular weight was found to be 60,000. The molecular weight determined from the electrophoretic mobility on several concentrations of gels by disc gel electrophoresis in the absence of SDS was found to be 120,000. These results indicate that monkey liver mitochondrial MAO exists as a dimer. Isoelectric focusing of the enzyme after solubilization with 0.1% Triton X-100 and 0.75% Triton X-100 and 0.75% Lubrol showed that it had a pI value near 6.5. Similar pI values were obtained for enzyme preparations solubilized with 0.75% Triton X-100 after treatment with phospholipase A or methylethylketone. These results suggest that the pI value of MAO in monkey liver mitochondria does not depend on the properties of detergents used to solubilize the enzyme preparation.     

DISSOCIATION,AGGREGATION OF SESAME α-GLOBULIN IN NONIONIC DETERGENT SOLUTION

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame α-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pK_Int value of the tyrosyl groups at 1 × 10^-3M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

Characterization of a phosphatidic acid phosphatase from rat brain cell membranes

We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [³²P] phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0. Mg²⁺ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca²⁺ at concentrations of 0.1 to 1 mmol/1. Zn²⁺ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/1 Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The K _m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V _max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M _r of 320000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D. Correspondence to: Ariane Hoer at the above address