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Racemic indan derivatives have been resolved by the hydrolysis of amide bonds using Corynebacterium ammoniagenes IFO12612 to produce (S)-amine and (R)-amides. In the kinetic resolution of 1 (N-12-(6-methoxy-indan-1-yl)ethyl]acetamide), it was possible to run the reaction to 44% conversion on a 10-g scale, obtaining (S)-amine 4 ((S)-2-(6-methoxy-indan-1-yl)ethylamine) at >99% enantiomeric excess (ee) and (R)-1 at 98% ee.
The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.
Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.
The biogenic amines are low-weight organic bases that exhibit variable biological activity. Approximately 30 vasoactive and psycoactive amines have been found in foodstuffs. Histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermine, and spermidine are examples of biogenic amines detectable in meat and its derivatives. These amines occur naturally in animals, plants, and microorganisms owing to their natural metabolic processes, and are usually formed by decarboxylation of amino acids.Histamine, 5-imidazol-ethylamine (MW 111), is produced by oxidative decarboxylation of histidine, and decarboxylase-positive microorganisms are able to produce it as a byproduct of their action on host tissues. This process, associated with enterobacteria such as Escherichia, Salmonella, Clostridium, and Bacillus, can be fast if the meat storage conditions are inadequate.Additionally, because of its nonvolatile behavior, histamine may bestow toxicity on the product even before it is considered decayed or organoleptically unacceptable. Secondary amines such as piperidine and pyrrolidine can be produced in the catabolism of histamine, and they, in turn, are precursors of nitrosamines, substances with known carcinogenic activity. Some cases of histamine toxicity have been reported.
This paper deals with rapid methods for identification of 50 yeast species frequently isolated from foods and fodders that underwent a lactic acid fermentation. However, many yeast species present in olive brine, alpechin, and other olive products were not treated. The methods required for identification include light microscopy, physiological growth tests (ID32C system of BioMérieux), assimilation of nitrate and of ethylamine as sole nitrogen sources, vitamin requirement, and maximum growth temperature. An identification key to treated yeast species is provided. In another table characteristics of all yeast species treated are listed.
A photoaffinity probe, 2-(4-phosphopentamanniminophenyl)ethyl-(4-azido) salicylamide, was prepared for photolabelling of the mannose 6-phosphate/insulin-like growth factor II receptor. The probe was synthesized from phosphopentamannan and 2-(4-aminophenyl)ethylamine followed by coupling with photosensitive 4-azidosalicylic acid. The 125I-labelled probe was reacted with receptor purified from rat liver under irradiation with ultraviolet light. Fluorography of the reaction product on a polyacrylamide gel effectively detected the receptor coupled to the photoprobe as a molecular mass of 250 kDa. Specific binding of the probe or of radiolabelled insulin-like growth factor II was not inhibited by the growth factor or by mannose 6-phosphate, confirming different binding sites to the receptor between the ligands.
The submandibular gland in situ was perfused with blood through the glandular artery at constant pressure in anesthetized dogs, and all drugs were administered intra-arterially. During infusion of metiamide, histamine and 2-(2-pyridyl)ethylamine (PEA) produced salivary and vasodilator responses consisting of an early and a late component. The dose-response curves for respective components of the salivary and vasodilator responses to PEA were parallel with the corresponding curves for histamine and in producing these responses PEA was about 40 times less potent than histamine on a molar basis. During infusion of mepyramine, histamine and dimaprit produced only the early vasodilator response. The dose-vasodilator response curves to histamine and dimaprit were parallel, and dimaprit was about 750 times less potent than histamine on a molar basis. The present results support the conclusion obtained in a previous study that neuronal histamine receptors mediating the whole salivary and the late vasodilator response are exclusively of the H1-type and vascular histamine receptors mediating the early vasodilator response consist of both H1-and H2-type although the former is predominant.
The role of structural fractions of dissolved organic matter (DOM) from wastewater in the sorption process of hydrophobic organic compounds is still not clear. In this study, DOM from two wastewater treatment plants (Lachish and Netanya, Israel) was fractionated to hydrophobic acid (HoA) and hydrophobic neutral (HoN) fractions. The fractions were characterized and their sorptive capabilities for s-triazine herbicides and polycyclic aromatic hydrocarbons (PAHs) were studied. For all sorbates, the binding to the HoN fractions was much higher than to HoA fractions. The HoA fractions were more polar than the HoN fractions, containing a higher level of carboxylic functionalities. However the higher binding coefficients of atrazine (2-chloro-4-ethylamine-6-isopropylamino-s-triazine) and ametryn [2-(ethylamino)-4-isopropylamino-6-methyl-thio-s-triazine] obtained for the HoN fractions suggest that their sorption is governed by hydrophobic-like interactions rather than H bonding. The values of binding coefficients of PAHs measured for the HoN fractions were within the range reported for humic acids and much higher than other fractions, suggesting that this fraction plays an important role in the overall sorption of these compounds by DOM. Higher sorption coefficients were measured for the Netanya DOM sample containing higher level of hydrophobic fractions (HoA + HoN) than the Lachish DOM, suggesting that the sorption of hydrophobic organic compounds by DOM is governed by the level of these structural substances. The evaluation of mobility of organic pollutants by wastewater irrigation requires not only assessment of the total carbon concentration but also, more importantly, the content of the hydrophobic fractions.
The purpose of this study was to propose a new method to predict in vivo drug-drug interactions (DDIs) for a high clearance drug from in vitro data. As the high clearance drug, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride) was used. First, approach based on I(u)/K(i) value was used for the prediction of DDIs between NE-100 and concomitant drugs. When the K(i) values (K(i-cal)) obtained from the microtiter plate (MTP) assay and the reported K(i) values (K(i-rep)) for these drugs were used to predict increases at levels of NE-100 AUC(oral) (AUC(oral) ratio), the AUC(oral) ratios from the I(u)/K(i-cal) correlated with those from the I(u)/K(i-rep). This result suggests that the K(i-cal) from the MTP assay can be used for prediction of DDIs instead of the K(i-rep) value. Second, a new approach combining the inhibition rate (R) calculated from the MTP assay and two physiological models was used to predict DDIs. When the AUC(oral) ratios of NE-100 by various drugs were predicted using the R value and the well-stirred model, the ratios were similar to those predicted using the I(u)/K(i). However, after co-administration of drugs such as quinidine, propafenone and thioridazine (potent inhibitors of CYP2D6), the NE-100 AUC(oral) ratios predicted from the dispersion model was much greater than those from well-stirred model. This result shows that application of the dispersion model to the prediction method using the R value might sensitively and precisely predict the increased levels of AUC(oral) by DDIs for high clearance drug, compared with the prediction method using I(u)/K(i) value.
Pale, soft, exudative (PSE) pork occurs, for the most part, from environmental stress on the pig. Amino acid intake may be related to stress susceptibility through hormone and neurotransmitter induction. Two experiments were conducted to determine whether supplementation of 5 g of tryptophan (TRP) or 10 g of tyrosine (TYR) per kilogram of a 14% CP diet would alter the response of pigs to stress as measured by hypothalamic neurotransmitter concentrations and incidence of PSE. Twenty-four (Exp. 1) and 36 (Exp. 2) 92-kg pigs were offered one of three diets: control, TRP-, or TYR-supplemented for 5 d before slaughter. Dietary TRP or TYR supplementation in Exp. 1. doubled (P less than .05) plasma TRP and TYR concentrations, respectively, and increased (P less than .05) 5-hydroxytryptamine, dihydroxyphenyl ethylamine, dihydroxyphenyl acetic acid, and homovanillic acid concentrations in the hypothalamus. Pigs that exhibited stress at slaughter had lower (P less than .05) hypothalamic concentrations of epinephrine, norepinephrine, and 5-hydroxytryptamine. In Exp. 2, pigs were trucked 55 km to a commercial meat packing facility and slaughtered without a rest period. This handling procedure was designed to invoke a high incidence of PSE pork and thus be a strong test of treatments. Supplemental dietary amino acids seemed to alter the frequency distribution of the severity of PSE pork. These data indicate that dietary manipulation of amino acid precursors of neurotransmitters may offer a practical means of reducing stress response in swine.
1. The hepatic microsomal metabolism of N-phenyl-2-naphthylamine (P2NA), an industrial antioxidant and suspected carcinogen, was studied in seven mammalian species to determine if N-dephenylation, reported to occur in vivo, could be demonstrated in vitro and if macromolecular binding occurs without an obligatory N-dephenylation. 2. The rate of hepatic microsomal metabolism decreased in the order: hamster much greater than mouse greater than rat congruent to monkey congruent to dog greater than human congruent to pig. Metabolism was increased by pretreatment with 3-methycholanthrene or phenobarbital and decreased by carbon monoxide or 2-[2,4-dichloro-6-phenyl)phenoxy]-ethylamine, which indicated the involvement of cytochrome P-450 rather than the flavoprotein mixed-function oxidase. 3. All seven species produced two major metabolites identified as 6-hydroxy-P2NA and 4-'hydroxy-P2NA. The carcinogen 2-naphthylamine was not detected in the microsomal incubations. 4. Incubations in the presence of 18O2 indicated that the oxygen incorporated in the products came from molecular oxygen. 5. There was a time-dependent linear increase in covalent binding of P2NA to microsomal protein and the extent of binding approximately paralleled the rate of metabolism. Thus, macromolecular binding of this aromatic amine appears to occur without an obligatory N-dephenylation and may be due to the metabolic formation of epoxides.
Fusogenic peptides derived from viral coat proteins cause perturbation of the endosomal membrane and are often used to improve the transfection efficiency of non-viral vectors in vitro. However, fusogenic peptides have limited potential for use in vivo due to their inherent immunogenicity. Totally synthetic polymers that are endosomolytic should circumvent this problem and could be useful as components of non-viral delivery systems as long as they do not immediately localise in the liver after intravenous (i.v.) injection. Linear poly(amidoamine) polymers (PAAs) having amido- and tertiary amino-groups along the main polymer undergo pH-dependent conformational change and thus provide an ideal opportunity for design of polymers that display membrane activity at low pH. Here we describe four PAAs, ISA 1 (Mn = 6900 Da) and ISA 23 (Mn = 10,500 Da) and their analogues ISA 4 and ISA 22 (Mn approximately 8000 Da) containing approximately 1 mol% 2-p-hydroxyphenyl ethylamine to allow radioiodination and thus monitoring of their biodistribution. In vitro cytotoxicity was assessed by MTT assay after incubation of PAAs with B16F10 and Mewo cell lines. The IC50 values observed for all PAAs were > 2 mg/mL in comparison with poly(L-lysine) which displayed an IC50 in the range 0.01-0.1 mg/mL. At pH 7.4 none of the PAAs studied was haemolytic at 1 h at concentrations below 3 mg/mL. PAAs were subsequently incubated with rat red blood cells for 24 h (1 mg/mL) at different pHs. In contrast to poly(L-lysine) which was haemolytic at pH 7.4, 6.5 and 5.5, none of the PAAs was lytic at pH 7.4, but they became membrane active at lower pH (approximately 45% for ISA 4, 50% for ISA 22 and 90% for ISA 23). These observations were substantiated by SEM and confirm the pH-dependence of membrane activity. After i.v. injection to rats 125I-labelled ISA 4 was immediately taken up by the liver (> 80% recovered dose at 1 h) whereas 125I-labelled ISA 22 was not (liver uptake was < 10% recovered dose at 5 h). Furthermore, biodistribution studies in mice bearing subcutaneous B16F10 melanoma showed that 125I-labelled ISA 22 was still accumulating in tumour tissue after 5 h (2.5% dose/g). PAAs have potential as endosomolytic agents and quantitation of the endosome to cytoplasm transfer is warranted after i.v. administration.
This paper describes the synthesis of 5-[(3-bromo-2,6-dimethoxybenzamido)-methyl]-5-hydroxy-2-pyrrolidon e (3) and its 1-ethyl analogue 2, two urinary metabolites of the dopamine D-2 antagonist remoxipride [1, (S)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide]. Two synthetic schemes leading to a common intermediate, 5-benzamido-4-oxopentanoic acid 4, were developed. This key intermediate permits conversion into either metabolite. Reaction of 4 with isobutyl chloroformate furnished a mixed carbonic anhydride, which upon treatment with ethylamine or ammonia gave the 4-oxopentanamides 5 and 6, respectively. Ring-closure afforded the corresponding 5-hydroxy-2-pyrrolidones 2 and 3.
The synthesis of two analogues of kainic acid (KA) incorporating photo-activatible moieties attached either on the gamma-carboxy function (gamma-amide 1) or the isopropenyl side-chain (amide 2) is described. The synthesis of the former amide involves coupling of N-(tert-butoxycarbonyl)-protected alpha-diphenylmethyl kainate with 2-(4-azidobenzamido)ethylamine (5) followed by trifluoroacetic acid mediated complete deprotection. Amide 2 was synthesized by palladium-mediated allylic amination, with 4,4'-dimethoxybenzhydrylamine (DMBA), of N-(9-fluorenylmethoxycarbonyl)-protected dimethyl kainate, followed by splitting the DMB-group with formic acid, coupling with N-hydroxysuccinimidoyl 4-azidobenzoate and finally complete deprotection by saponification. Preliminary pharmacological studies in chicken brain membranes showed that amide 2 is a stronger inhibitor of [3H]KA binding on chicken cerebellar membranes than is amide 1 and that amide 2 has specificity only for the cerebellar, as opposed to the telencephalon, type of non-NMDA binding sites.
Amino acid esters can destroy intracellular as well as isolated amastigotes of Leishmania mexicana amazonensis. In the present study we examined, using a tetrazolium reduction assay, the toxicity of the esters for amastigotes isolated from mouse lesions. Parasite killing by the "prototype" compound L-leucine methyl ester at 1 mM concentration and at pH 7.3 took place within 15-30 min. Time-lapse cinematographic observations showed that the amastigotes rounded up and became less phase-dense before they rapidly broke down. Ammonium chloride, ethylamine or monensin, known to raise the intracellular pH, reduced the sensitivity of the amastigotes to L-Leu-OMe. This finding suggests that an acidified compartment is involved in the destruction of the parasites. The leishmanicidal activity of a series of L-amino acid esters was also investigated. The ED50 (concentration for half maximal effect) for methyl esters was (in mM): Leu (0.62), Trp (0.96), Met (1.13), Glu (2.0), Phe (2.5), and Tyr (3.8). In contrast, the methyl esters of Ile, Val, Ala, beta Ala, Gly, Ser, His, and Pro were either inactive or weakly active at 15 mM. Benzyl esters were more active than their methyl homologs: the ED50 of the benzyl esters of Leu, Val, Ile, Gly, Ala, beta Ala, and Pro were, respectively, 0.07, 0.20, 0.22, 0.88, 1.5, 2.3, and 6.7 mM. Ranks of leishmanicidal activity may reflect differences in the rates of ester uptake and trapping by the amastigotes, in the specificity of the relevant hydrolytic enzyme(s), in the accumulation and metabolic fate of the released amino acids, or in the toxicity of the amino acid or alcohol released within the amastigotes.
Biogenic amines play an important physiological role in mammals, and high amounts of some exogenous amines in human diet may contribute to a wide variety of toxic effects. These amines are commonly found in many foodstuffs, particularly in fermented products such as cheese, meat products, beer, wine, and ciders. Here, the level of biogenic amines in some natural ciders was examined. Twenty-four samples of cider purchased from commercial sources were analyzed by reverse-phase high-performance liquid chromatography and fluorescence detection after precolumn derivatization with o-phthaldialdehyde. Amine levels were variable, ranging from not detected to 23 mg/liter. The average level of total biogenic amines in ciders was 5.94 +/- 8.42 mg/liter. Putrescine, histamine, and tyramine were the prevailing amines being present in 50.0, 37.5, and 33.3% of the ciders studied; very small amounts of ethylamine and phenylethylamine were observed in only one sample. Other cider parameters were analyzed to determine whether they affect the biogenic amine content in ciders, and the results were evaluated by applying cluster analysis and principal component analysis. Ciders that showed lower glycerol contents and higher amounts of 1,3-propanediol had much higher levels of histamine, tyramine, and putrescine, suggesting a high activity of lactic acid bacteria during cider making and thus the need for effective control of lactic acid bacteria.
When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of mitomycin analogs described as Type I and Type II are produced. Type I analogs are related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogs also contain the same substitutions at C7 but the conformation of the mitosane ring is related to mitomycin B having an OH at positions C9a and a methyl substituted aziridine. The products obtained from the supplementation of the medium with methylamine, ethylamine, propylamine, propargylamine and 2-methylallylamine were isolated and characterized. In all cases the Type I analogs are more active in a prophage induction test and against L1210 lymphatic leukemia in mice. A number of other amines have been tested and shown to yield new products that have not yet been isolated. No secondary amines are incorporated.
3-Amino-2-hydroxyvaleric acid was prepared, and separated into its diastereomers. The relative stereochemistry was determined by 1H NMR in their oxazolidone derivatives. The threo-isomer was resolved by (S)-1-(1-naphthyl) ethylamine in the N-(p-methoxybenzyloxycarbonyl) derivative. The absolute configuration of (--)-threo-3-(p-methoxybenzyloxycarbonyl)amino-2-hydroxyvaleric acid was confirmed to be 2R, 3S. The absolute configuration of 3-amino-2- oxovaleric acid in poststatin was confirmed to be S by comparison of the four stereoisomers of methyl N, O-bis(3,5-dinitrobenzoyl)-3-amino-2-hydroxyvalerate derived from 3-amino-2-hydroxyvaleric acid and that derived from 3-amino-2-oxovaleryl moiety of poststatin by means of HPLC with chiral column.