N-alkylated nitrogen-in-the-ring sugars: conformational basis of inhibition of glycosidases and HIV-1 replication

The conformations of nitrogen-in-the-ring sugars and their N-alkyl derivatives were studied from 1H NMR analyses, mainly using 3J(H,H) coupling constants and quantitative NOE experiments. No significant difference was seen in the ring conformation of 1-deoxynojirimycin (1), N-methyl-1-deoxynojirimycin (2), and N-butyl-1-deoxynojirimycin (3). However, it was shown that the C6 OH group in 1 is predominantly equatorial to the piperidine ring, while that in 2 or 3 is predominantly axial, and its N-alkyl group is oriented equatorially. In the furanose analogues 1,4-dideoxy-1,4-imino-D-arabinitol (4) and its N-methyl (5) and N-butyl (6) derivatives, the five-membered ring conformation differed significantly by the presence or absence of the N-substituted group and the length of the N-alkyl chain. Compound 3 reduced its inhibitory effect on almost all glycosidases, resulting in an extremely specific inhibitor for processing alpha-glucosidase I since N-alkylation of 1 is known to enhance both the potency and specificity of this enzyme in vitro and in vivo. This preferred (C6 OH axial) conformation in 2 and 3 appears to be responsible for their strong alpha-glucosidase I activity. Compound 4 is a good inhibitor of intestinal alpha-glucohydrolases, alpha-glucosidase II, and Golgi alpha-mannosidases I and II, but its N-alkyl derivatives 5 and 6 markedly decreased inhibitory potential for all enzymes tested. In the case of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP, 7), which is a potent beta-galactosidase inhibitor, its N-methyl (8) and N-butyl (9) derivatives completely lost potency toward beta-galactosidase as well. N-Alkylation of compounds 4 and 7, known well as potent yeast alpha-glucosidase inhibitors, resulted in a serious loss of inhibitory activity toward yeast alpha-glucohydrolases. Activity of these nine analogues against HIV-1 replication was determined, based on the inhibition of virus-induced cytopathogenicity in MT-4 and MOLT-4 cells. Compounds 2 and 3, which are better inhibitors of alpha-glucosidase I than 1, proved active with EC50 values of 69 and 49 micrograms/mL in MT-4 cells and 100 and 37 micrograms/mL in MOLT-4 cells, respectively, while none of the furanose analogues exhibited any inhibitory effects on HIV-1. The change in potency and specificity of bioactivity by N-alkylation of nitrogen-in-the-ring sugars appears to be correlated with their conformational change.     

Experimental infection of domestic cats with Bartonella henselae by inoculation of Ctenocephalides felis (Siphonaptera: Pulicidae) feces

Aqueous methanol extracts from the bulbs of Hyacinthusorientalis were subjected to various ion-exchange column chromatographic steps to give 2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine (DMDP) (1), 2,5-dideoxy-2,5-imino-dl-glycero-d-manno-heptitol (homoDMDP) (2), 2,5-imino-2,5,6-trideoxy-d-manno-heptitol (6-deoxy-homoDMDP) (3), 2,5-imino-2,5,6-trideoxy-d-gulo-heptitol (4), 1-deoxynojirimycin (5), 1-deoxymannojirimycin (6), alpha-homonojirimycin (7), beta-homonojirimycin (8), alpha-homomannojirimycin (9), beta-homomannojirimycin (10), and 7-O-beta-d-glucopyranosyl-alpha-homonojirimycin (MDL 25,637) (11). The structures of the new natural products 3 and 4 were determined by spectroscopic analysis, including extensive 1D and 2D NMR studies. Compound 2 was found to be a potent inhibitor of bacterial beta-glucosidase, mammalian beta-galactosidases, and mammalian trehalases, while 3 was a potent inhibitor of rice alpha-glucosidase and rat intestinal maltase. Compound 4 was observed to be a good inhibitor of alpha-l-fucosidase.     

Nectrisine is a potent inhibitor of alpha-glucosidases, demonstrating activities similarly at enzyme and cellular levels

Nectrisine, discovered as an immunomodulator, was found to inhibit alpha-glucosidase, alpha- and beta-mannosidases, beta-glucosidase and beta-N-acetylglucosaminidase, in that order of inhibition strength. Beta-Galactosidase, alpha-fucosidase, and neuraminidase were insensitive to this antibiotic. Also sensitive was the trimming glucosidase I which participates in the first step of modifying N-glycosidic oligosaccharide. Nectrisine demonstrated an inhibitory effect at the cellular level as strong as expected based on its action at enzyme levels; castanospermine and 1-deoxynojirimycin did not. Nectrisine and castanospermine suppressed syncytium formation and hemolytic activity in Newcastle disease virus (NDV)-infected BHK cells, without blocking the synthesis and cell-surface expression of HANA glycoprotein of NDV.     

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography

Two kojibiose-type pseudo-disaccharides and a trisaccharide, containing a 5-amino-1,2,3,4-cyclopentanetetrol derivative or valienamine, linked by way of nitrogen bridges to the sugar residues, have been designed and synthesized as processing alpha-glucosidase I inhibitors. Synthesis of the pseudo-disaccharides was carried out starting from the coupling products of the sugar isothiocyanates and an aminocyclitol, respectively, by cyclization with mercury(II) oxide to the cyclic isoureas and subsequent deprotection. Pseudokojibiose was prepared in a poor yield by reaction of a protected valienamine and a sugar epoxide, followed by deprotection. Although the pseudooligosaccharides are all strong inhibitors of alpha-glucosidase (baker's yeast), they did not have any inhibitory potency against either sucrase isomaltase (rat intestine) or processing alpha-glucosidase (rat liver microsomes).     

N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, markedly reduced infarct size in rabbit hearts

BACKGROUND: N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, reduces the glycogenolytic rate by inhibiting the alpha-1,6-glucosidase of glycogen-debranching enzyme in the liver, in addition to possessing an antihyperglycemic action by blocking alpha-1,4-glucosidase in the intestine. Because the reduction of the glycogenolytic rate may be one of the mechanisms of myocardial protection in ischemic preconditioning, the compounds inhibiting myocardial alpha-1,6-glucosidase may be protective against ischemic damage. Thus, we investigated whether MOR-14 could inhibit alpha-1,6-glucosidase and reduce the infarct size in rabbit hearts without collateral circulation. METHODS AND RESULTS: MOR-14 dose-dependently decreased the alpha-1,6-glucosidase activity in rabbit heart extract. A tracer study demonstrated the myocardial uptake of a considerable amount of MOR-14 sufficient to fully inhibit alpha-1,6-glucosidase. To assess the infarct size-reducing effect of MOR-14, 54 rabbits were subjected to 30-minute coronary occlusion followed by 48-hour reperfusion. Preischemic treatment with 25, 50, and 100 mg/kg of MOR-14 dose-dependently reduced the infarct size (to 26+/-4%, 19+/-3%, and 14+/-2% of the area at risk, respectively), compared with the saline control (45+/-5%) without altering the blood pressure or heart rate. Another 40 rabbits given 100 mg of MOR-14 or saline 10 minutes before ischemia were euthanized at 10 or 30 minutes of ischemia for biochemical analysis. MOR-14 decreased the alpha-1,6-glucosidase activity to approximately 20% in vivo, reduced the glycogen breakdown, and attenuated the lactate accumulation at both 10 and 30 minutes of ischemia. CONCLUSIONS: Preischemic treatment with MOR-14 preserved glycogen, attenuated the accumulation of lactate, and reduced the myocardial infarct size by 69%. This cardioprotective effect was independent of changes of blood pressure and heart rate or regional blood flow. It may be associated with alpha-1,6-glucosidase inhibition, because MOR-14 markedly decreased the alpha-1,6-glucosidase activity in the heart.     

Inhibition of thrombin-induced contractile responses by protein kinase inhibitors in porcine pulmonary arteries

The clotting enzyme thrombin is known to cause receptor-mediated contractile effects in isolated blood vessels. In the present studies the influence of protein kinase inhibitors on the contractile response of porcine pulmonary arteries to thrombin (3 U/ml) was investigated. Endothelium-denuded rings (2-3 mm) from small arteries were placed in organ baths for isometric tension recording. The vessels were preincubated for 30 min with the inhibitors before inducing contractions. In the presence of the protein kinase C (PKC)-inhibitors staurosporine, BIM I (bisindolyl-maleimide I), chelerythrine and Ro 31-8220 (1 microM each), the contractile responses to the PKC activator phorbol 12,13-dibutyrate (PDBu; 50 nM) were diminished by 70-100%. However, for inhibition of thrombin-induced contractions generally higher concentrations of the inhibitors were required. Only staurosporine at 1 microM inhibited the thrombin effect by about 75%. The tyrosine kinase inhibitor erbstatin (30 microM) did not significantly alter the thrombin effect, whereas genistein at 10 microM caused a significant inhibition of contractile responses to both thrombin and PGF2alpha. At 100 microM genistein also inhibited the contractile effects of PdBu and KCl. These studies suggest that activation of both PKC and non-receptor tyrosine kinases seems to be involved in the signal transduction pathways of thrombin-induced contractile effects in isolated vessels.     

Novel peptidyl phosphorus derivatives as inhibitors of human calpain I

Dipeptidyl phosphorus compounds were synthesized as potential bioisosteric mimics of peptide alpha-ketoesters and alpha-ketoacids. alpha-Ketophosphonate Cbz-Leu-Leu-P(O)(OCH3)2 (1b), containing an alpha-ketoester bioisostere, inhibits human calpain I with an IC50 = 0.43 microM. The potency of 1b compares very favorably with that of alpha-ketoester Cbz-Leu-Leu-CO2Et (IC50 = 0.60 microM). Monomethyl ketophosphonate Cbz-Leu-Leu-P(O)(OH)(OCH3) (1a, IC50 = 5.2 microM), an alpha-ketoacid mimic, is less potent. Dibutyl and dibenzyl alpha-ketophosphonates 1c,e,f are much less potent calpain inhibitors than dimethyl alpha-ketophosphonate 1b. alpha-Ketophosphinate 1g (IC50 = 0.37 microM) and alpha-ketophosphine oxide 1h (IC50 = 0.35 microM) are also potent calpain inhibitors.     

Processing glycosidases of Saccharomyces cerevisiae

The properties of the N-glycan processing glycosidases located in the endoplasmic reticulum of Saccharomyces cerevisiae are described. alpha-Glucosidase I encoded by CWH41 cleaves the terminal alpha1, 2-linked glucose and alpha-glucosidase II encoded by ROT2 removes the two alpha1,3-linked glucose residues from the Glc3Man9GlcNAc2 oligosaccharide precursor while the alpha1,2-mannosidase encoded by MNS1 removes one specific mannose to form a single isomer of Man8GlcNAc2. Although trimming by these glycosidases is not essential for the formation of N-glycan outer chains, recent studies on mutants lacking these enzymes indicate that alpha-glucosidases I and II play an indirect role in cell wall beta1,6-glucan formation and that the alpha1,2-mannosidase is involved in endoplasmic reticulum quality control. Detailed structure-function studies of recombinant yeast alpha1,2-mannosidase are described that serve as a model for other members of this enzyme family that has been conserved through eukaryotic evolution.     

Substrates and inhibitors of human T-cell leukemia virus type I protease

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.     

Mechanism of alpha-2 adrenergic modulation of canine cardiac Purkinje action potential

We reported recently that stimulation of postjunctional alpha-2 adrenergic receptors prolongs the action potential durations (APD) of isolated canine Purkinje fibers. With standard microelectrode techniques, we examined the ionic mechanism through which alpha-2 adrenergic stimulation prolonged Purkinje APD, by measuring the effects of inhibitors of the various plateau currents on the alpha-2-mediated prolongation of APD. The alpha-2-specific agonist UK 14,304 (0.1 microM) prolonged the Purkinje APD at 50% repolarization and the APD at 90% repolarization, and these effects were inhibited by yohimbine (0.1 microM). The Purkinje APD at 50% repolarization and the APD at 90% repolarization were prolonged significantly with the transient outward potassium current inhibitor 4-aminopyridine (1 mM), the rapid component of delayed rectifier potassium current inhibitor d-sotalol (10 microM), the slow component of delayed rectifier potassium current inhibitor indapamide (0.1 microM) and the chloride current inhibitor mefenamic acid (10 nM) and were shortened significantly with the calcium current inhibitor nifedipine (0.3 microM). Prolongation of Purkinje APD at 50% repolarization and APD at 90% repolarization by UK 14,304 remained intact in the presence of d-sotalol, indapamide, mefenamic acid and nifedipine. All of these UK 14,304 effects were significantly reversed by yohimbine. Only in the presence of 4-aminopyridine did UK 14,304 fail to prolong Purkinje APD. The phase 1 magnitudes of Purkinje action potentials were also significantly inhibited by UK 14,304. This effect was completely abolished only in the presence of 4-aminopyridine. These results suggest that inhibition of the 4-aminopyridine-sensitive transient outward potassium current is the major ionic mechanism by which alpha-2 adrenergic stimulation prolongs Purkinje APD.     

Some 1-[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17 alpha-hydroxylase: 17, 20-lyase (P450 17) and their specificity patterns

Four 1-[(benzofuran-2-yl)methyl]imidazoles (1-4) have been evaluated as in-vitro inhibitors of human testicular and bovine adrenal microsomal 17 alpha-hydroxylase: 17,20-lyase (P450 17) as potential anti-prostatic agents. Their specificity towards other steroidogenic and liver enzymes has been compared with that of ketoconazole. All four compounds were inhibitors of the testicular enzyme (2, IC50 (concentration resulting in 50% inhibition) 0.185 microM; 4, IC50 0.18 microM) but less potent than ketoconazole (IC50 0.03 microM). Towards bovine adrenal enzyme 2 and 4 were 35- and 31-fold more potent than ketoconazole (IC50 = 39.8 microM). Compound 2 is a useful lead compound but although less potent than ketoconazole towards P450SCC and P450 11 beta, but not P450C21, at the enhanced dose required for equivalent effects in-vivo on P450 17 it is likely that cortisol and aldosterone production will be affected to a greater extent than with ketoconazole.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Rapid reversed-phase high-performance liquid chromatography method for quantitation, at high pH, of the recombinant apolipoprotein A-IMilano in Escherichia coli fermentation broth

In an effort to develop potent agents for reducing the levels of the active estrogen, estradiol, we developed a new category of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1 inhibitors. The compounds described possess a butyl methyl alkylamide side chain linked to the C6 position of estradiol by a thioether. With a series of epimeric mixtures, an optimal side-chain length of five methylene groups (between the amide group and steroid part) was first determined. Thereafter, both C6 epimers of optimized mixture were obtained after high-pressure liquid chromatography separation. 1H and 13C NMR experiments were performed to confirm the stereochemistry of each epimer. The 6beta-orientation of the side-chain was found to be crucial for enzymatic inhibition. Indeed, for the optimized side-chain length, the compound with a beta-orientation (5: N-butyl,N-methyl 7-(3',17'beta-dihydroxy-1',3',5'( 10')-estratriene-6'beta-yl)-7-thiaheptanamide) was 70-fold more potent than the 6alpha-analog. Compound 5 did not inactivate 17beta-HSD type 1, suggesting a reversible inhibitor. In addition, it was found to be a more potent inhibitor than the substrate estrone itself or a panel of three known inhibitors.     

10-Formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): a potent inhibitor of glycinamide ribonucleotide transformylase

The synthesis of 10-formyl-5,8,10-trideazafolic acid (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a Ki of 0.26 +/- 0.05 microM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with Ki of 7.6 +/- 1.5 microM.     

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.     

Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase

The rational design, synthesis, and biological activity of farnesyl diphosphate (FPP)-based inhibitors of the enzyme Ras farnesyl protein transferase (FPT) is described. Compound 3, wherein a beta-carboxylic phosphonic acid type pyrophosphate (PP) surrogate is connected to the hydrophobic farnesyl group by an amide linker, was found to be a potent (I50(FPT) = 75 nM) and selective inhibitor of FPT, as evidenced by its inferior activity against squalene synthetase (I50(SS) = 516 microM) and mevalonate kinase (I50(MK) = > 200 microM). A systematic structure-activity relationship study involving modifications of the farnesyl group, the amide linker, and the PP surrogate of 3 was undertaken. Both the carboxylic and phosphonic acid groups of the beta-carboxylic phosphonic acid PP surrogate are essential for activity, since deletion of either group results in 50-2600-fold loss in activity (6-9, I50 = 4.6-220 microM). The farnesyl group also displays very stringent requirements and does not tolerate one carbon homologation (12, I50 = 17.7 microM), substitution by a dodecyl fragment (14, I50 = 9 microM), or introduction of an extra methyl group at the allylic position (18, I50 = 55 microM). Modifications around the amide linker group of 3 were more forgiving, as evidenced by the activity of N-methyl analog (21, I50 = 0.53 microM), the one carbon atom shorter farnesoic acid-derived retroamide analog (32, I50 = 250 nM), and the exact retroamide analog (49, I50 = 50 nM). FPP analogs such as 3, 32, and 49 are novel, potent, selective, small-sized, nonpeptidic inhibitors of FPT that may find utility as antitumor agents.     

Oxidation products of hyperforin from Hypericum perforatum

The isolation of two oxidation products of hyperforin from the aerial parts of Hypericum perforatum and their structure determination by means of 2D NMR methods is reported. The products had the same 1-(2-methyl-1-oxopropyl)-2,12-dioxo-3,10 beta-bis(3-methyl-2-butenyl)-11 beta-methyl-11 alpha-(4-methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0(4,8)]-3-dodec ene skeleton. In addition, one of them, with the same number of carbons as hyperforin (C35H52O5), contained a 1-methyl-l-hydroxyethyl group in the 6 beta-position, whereas the other compound (a hemiacetal, C32H46O5), presumably a degradation product of hyperforin, exhibited a 6-hydroxy function. The latter was an inseparable mixture of 6 alpha- and 6 beta-hydroxy epimers undergoing (according to phase sensitive NOESY) mutual interconversion.     

Structure, properties, and tissue localization of apoplastic alpha-glucosidase in crucifers

Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.     

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and diindolylmethane (DIM) is the major acid-catalyzed condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah-responsive T47D human breast cancer cells, I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 microM, respectively. A 1 nM concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells, and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31 microM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.