Organic properties of sediments and amino acids in iterstitial waters of ODP Holes 111-677A and 111-678B

The basement at Ocean Drilling Program (ODP) Sites 677 and 678 originated from the Galapagos spreading center of the Costa Rica Rift and has moved about 200 km over the last 6 m.y. (Fig. 1) (Shipboard Scientific Party, 1987, 1988; Scientific Drilling Party, 1987). Sediments about 300 m thick cover basement so young that basal sediments at Sites 677 and 678 have been reheated up to 60?-70?C at Site 677 and altered to limestone and/or chert (Shipboard Scientific Party, 1988). Sediments from both sites indicate (1) a high sedimentation rate (about 48 m/m.y.) and (2) biogenic silica and carbonate as the main constituents of sediments (Table 1) (Shipboard Scientific Party, 1988). Heatflow observations and measurements of interstitial water chemistry around the sites show that Site 677 is in a lower heatflow zone (166 mW/m**2; 1°12.14'N, 83°44.22'W) whereas Site 678 is located in a zone of higher heat flow (250 mW/m**2; 1°13.01'N, 83°43.39'W) (Langseth et al., 1988; Shipboard Scientific Party, 1988). In the flank hydrothermal systems, circulating solution is moving upward through the sedimentary column in zones of higher heat flow while it is moving downward in zones of lower heat flow (Anderson and Skilbeck, 1981). The chemistry of the interstitial waters is modified by several processes such as (1) diagenetic reactions and (2) advective and (3) diffusive transports of dissolved constituents. Analyses of Ca2+ and Mg2+ in interstitial waters from Sites 677 and 678 show that their profiles are mainly controlled by advective transport (Shipboard Scientific Party, 1988). In contrast, the interstitial-water profiles for NH4+, Si, and PO4[3-] are highly affected by reactions in the sediments. Site 677 offers a good opportunity to investigate amino acids in the interstitial waters because sediments of similar compositions have been deposited at constant rates of sedimentation. There are few previous works on amino acid distributions in interstitial waters (Henrichs and Parrington, 1979; Michaelis et al., 1982; Henrichs et al., 1984; Henrichs and Farrington, 1987; Ishizuka et al., 1988). In this chapter, we report (1) Rock-Eval analysis and (2) the composition of total hydrolyzable and dissolved free amino acids (THAA and DFAA, respectively) in the interstitial waters. Our objectives are to discuss (1) the possible origin of organic materials, (2) the characteristics of THAA and DFAA, and (3) their relationships in interstitial waters.

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.

Enrichment, isolation and characterisation of ruminal bacteria that degrade non-protein amino acids from the tropical legume Acacia angustissima

Acacia angustissima has been proposed as a protein supplement in countries where low quality forages predominate. A number of non-protein amino acids have been identified in the leaves of A. angustissima and these have been linked to toxicity in ruminants. The non-protein amino acid 4-n-acetyl-2,4-diaminobutyric acid (ADAB) has been shown to be the major amino acid in the leaves of A. angustissima. The current study aimed to identify micro-organisms from the rumen environment capable of degrading ADAB by using a defined rumen-simulating media with an amino acid extract from A. angustissima. A mixed enrichment culture was obtained that exhibited substantial ADAB-degrading ability. Attempts to isolate an ADAB-degrading micro-organism were carried out, however no isolates were able to degrade ADAB in pure culture. This enrichment culture was also able to degrade the non-protein amino acids diaminobutyric acid (DABA) and diaminopropionic acid (DAPA) which have structural similarities to ADAB. Two isolates were obtained which could degrade DAPA. One isolate is a novel Grain-positive rod (strain LPLR3) which belongs to the Firmicutes and is not closely related to any previously isolated bacterium. The other isolate is strain LPSR1 which belongs to the Gammaproteobacteria and is closely related (99.93% similar) to Klebsiella pneumoniae subsp. ozaenae. The studies demonstrate that the rumen is a potential rich source of undiscovered micro-organisms which have novel capacities to degrade plant secondary compounds. (c) 2005 Elsevier B.V. All rights reserved.