Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: Mechanistic studies in embryonic cultured hippocampal neurones

Indirect evidence indicates that morphine-3-glucuronide (M3G) may contribute significantly to the neuro-excitatory side effects (myoclonus and allodynia) of large-dose systemic morphine. To gain insight into the mechanism underlying M3G' s excitatory behaviors, We used fluo-3 fluorescence digital imaging techniques to assess the acute effects of M3G (5-500 muM) on the cytosolic calcium concentration ([Ca2+](CYT)) in cultured embryonic hippocampal neurones. Acute (3 min) exposure of neurones to M3G evoked [Ca2+](CYT) transients that were typically either (a) transient oscillatory responses characterized by a rapid increase in [Ca2+](CYT) oscillation amplitude that was sustained for at least similar to30 s or (b) a sustained increase in [Ca2+](CYT) that slowly recovered to baseline. Naloxone-pretreatment decreased the proportion of M3G-responsive neurones by 10%-25%, implicating a predominantly non-opioidergic mechanism. Although the naloxone-insensitive M3G-induced increases in [Ca2+](CYT) were completely blocked by N-methyl-D-aspartic acid (NMDA) antagonists and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (alphaamino-3-hydroxy-5-methyl-4-isoxazolepropiordc acid/ kainate antagonist), CNQX did not block the large increase in [Ca2+](CYT) evoked by NMDA (as expected), confirming that N13G indirectly activates the NMDA receptor. Additionally, tetrodotoxin (Na+ channel blocker), baclofen (gamma-aminobutyric acid, agonist), MVIIC (P/Q-type calcium channel blocker), and nifedipine (L-type calcium channel blocker) all abolished M3G-induced increases in [Ca2+](CYT), suggesting that M3G may produce its neuro-excitatory effects by modulating neurotransmitter release. However, additional characterization is required.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Ileal digestibility of amino acids in feed ingredients for broilers.

To more precisely formulate feed and predict animal performance, it is important to base both the recommendations and feed formulations on digestible rather than total amino acid contents. Most published data on the digestibility of amino acids in feed ingredients for poultry are based on excreta digestibility. Ileal digestibility is an alternative and preferred approach to estimate amino acid availability in feed ingredients. Both methodologies are described and assessed. In addition, the differences between apparent and standardised (in which corrections are made for basal endogenous losses) digestible amino acid systems are discussed. The concept of a standardised digestibility system as a mean of overcoming the limitations of apparent digestibility estimates is proposed. In this context, different methodologies for the determination of basal endogenous amino acid losses are discussed. Although each methodology suffers from some limitations and published data on endogenous losses at the ileal level in growing poultry are limited, averaged data from repeated experiments using the 'enzymatically hydrolysed casein' method are considered as the best measure of basal losses. Standardised ileal amino acid digestibility values of 17 feed ingredients commonly used in broiler nutrition are presented including grains (barley, corn, sorghum, triticale, wheat), grain by-products (wheat middlings, rice pollard), plant protein sources (soybean meal, canola meal, corn gluten meal, cottonseed meal, lupins, peas/beans, sunflower meal), and animal by-products (feather meal, fish meal, meat and bone meal). This comprehensive set of the ileal amino acid digestibility of feed ingredients in broiler nutrition may serve as a basis for the establishment of the system in broiler feeding and for further research.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Apparent ileal digestibility of amino acids in dietary ingredients for broiler chickens

The apparent ileal digestibility coefficients of amino acids in 107 samples representing 22 food ingredients were determined using 6-week-old broiler chickens. The ingredients assayed included five cereals ( barley, maize, sorghum, triticale and wheat), two cereal by-products ( rice polishings and wheat middlings), four oilseed meals ( canola, cottonseed, soyabean and sunflower meals), full-fat canola, maize gluten meal, four grain legumes ( chickpeas, faba beans,field peas and lupins) and five animal protein sources ( blood, feather,fish, meat and meat and bone meals). The mean ileal digestibility coefficients of amino acids in wheat and maize were higher than those in sorghum, triticale and barley. However, variations observed in individual amino acid digestibilities among samples within cereal type were greater than those determined between cereals. Threonine and lysine were the least digestible indispensable amino acids in the five cereals evaluated. The most digestible indispensable amino acid was phenylalanine in wheat and, leucine in maize and sorghum. In the case of the wheat middlings and rice polishings, threonine was the least digestible indispensable amino acid and arginine was the best digested. In the oilseed meals assayed, amino acid digestibility was highest for soya-bean and sunflower meals, intermediate for canola meal and lowest for cottonseed meal. Ileal digestibility coefficients of amino acids in lupins were found to be slightly lower than those in soya-bean meal. The amino acid digestibilities of field peas, faba beans and chickpeas were considerably lower than those of lupins. Digestibility of arginine was the highest and that of threonine was the lowest of the indispensable amino acids in oilseed meals and grain legumes, except in cottonseed meal. Lysine was the least digestible amino acid in cottonseed meal. In the animal protein sources assayed, digestibility coefficients of amino acids in blood meal were high, intermediate in fish meal, and low in meat meal, meat and bone meal and feather meal. Variation in amino acid digestibility coefficients determined for blood meal samples was small. However, wide variations in amino acid digestibilities were observed for other animal protein sources, highlighting significant batch-to-batch differences. In particular, marked variations were determined for meat meal and meat and bone meal samples. Cystine was the least digested amino acid in animal protein meals, with the exception of blood meal in which isoleucine had the lowest digestibility. The limitations of using apparent digestibility values in diet formulations and the concept of the standardized digestibility system to overcome these limitations are discussed.

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) alpha-amylase inhibitor transgene

The nutritive value of transgenic peas expressing an a-amylase inhibitor (alpha-Ail) was evaluated with broiler chickens. The effects of feeding transgenic peas on the development of visceral organs associated with digestion and nutrient absorption were also examined. The chemical composition of the conventional and the transgenic peas used in this study were similar. In the two feeding trials, that were conducted normal and transgenic peas were incorporated into a maize-soybean diet at concentrations up to 500 g kg(-1). The diets were balanced to contain similar levels of apparent metabolisable energy (AME) and amino acids. In the first trial, the birds were fed the diets from 3 to 17days post-hatching and with levels of transgenic peas at 250 g kg(-1) or greater there was a significant reduction in body weight but an increase in feed intake resulting in deceased feed conversion efficiency. In the second trial, in which the birds were fed diets containing 300 g kg(-1) transgenic peas until 40 days of age, growth performance was significantly reduced. It was also demonstrated that the ileal starch digestibility coefficient (0.80 vs 0.42) was significantly reduced in the birds fed transgenic peas. Determination of AME and ileal digestibility of amino acids in 5-week-old broilers demonstrated a significant reduction in AME (12.12 vs 5.08 MJ kg(-1) DM) in the birds fed the transgenic peas. The AME value recorded for transgenic peas reflected the lower starch digestibility of this line. Real digestion of protein and amino acids was unaffected by treatment. Expression of a-Ail in peas did not appear to affect bird health or the utilisation of dietary protein. However, the significant reduction in ileal digestion of starch in transgenic peas does reduce the utility of this feedstuff in monogastric diets where efficient energy utilisation is required. (c) 2006 Society of Chemical Industry.