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.

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.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 Angstrom resolution, have unit-cell parameters a = b = 179.92, c = 185.82 Angstrom and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

Relationship of body condition score and blood urea and ammonia to pregnancy in Italian Mediterranean buffaloes

The relationship of body condition score ( BCS) and blood urea and ammonia to pregnancy outcome was examined in Italian Mediterranean Buffalo cows mated by AI. The study was conducted on 150 buffaloes at 145 +/- 83 days in milk that were fed a diet comprising 14.8% crude protein, 0.9 milk forage units . kg(-1) dry matter and a non- structural carbohydrate/ crude protein ratio of 2.14. The stage of the oestrous cycle was synchronised by the Ovsynch- TAI programme and blood urea and ammonia levels were assessed on the day of AI. Energy corrected milk ( ECM) production and BCS were recorded bi- weekly. The pregnancy risk was 46.7% and was slightly lower in buffaloes with BCS < 6.0 and BCS > 7.5. There were no significant differences in ECM, urea and ammonia between pregnant and non- pregnant buffaloes. However, pregnancy outcome was higher ( P = 0.02) in buffaloes with blood urea < 6.83 mmol . L-1. The likelihood of pregnancy for buffaloes with low urea blood level was 2.6 greater than for high urea level and exposure to a high urea level lowered the probability of pregnancy by about 0.25. The findings indicate that buffaloes are similar to cattle and increased blood levels of urea are associated with reduced fertility when animals are mated by AI.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Effects of phytase supplementation of diets with two tiers of nutrient specifications on growth performance and protein efficiency ratios of broiler chickens

in two feeding experiments male and mixed-sex broiler chicks were offered diets based on sorghum and a wheat-sorghum blend with two tiers of nutrient specifications, without and with microbial phytase (600 and 800 FTU/kg), from 7-25 and 1-42 days post-hatch, respectively. The nutrient specifications for protein, amino acids, energy density and phosphorus (P) of standard diets were reduced to formulate the modified diets on a least-cost basis. Calculated differences in nutrient specifications between standard and modified diets ranged from 14.3 to 17.1 g/kg crude protein, 0.24 to 0.40 MJ/kg apparent metabolisable energy (AME) and 1.06 to 1.20 g/kg available P. In both experiments, reduced nutrient specifications had a negative impact on growth rates and feed efficiency and phytase supplementation had a positive influence on growth performance and protein efficiency ratios (PER). Phytase addition to the less expensive, modified diets either partially or entirely compensated for reduced growth performance and, consequently, feed costs per kg of live weight gain were reduced. In Experiment 1, phytase increased (p<0.001) nitrogen-corrected AME (AMEn) from 15.39 to 15.89 MJ/kg dry matter. For nitrogen (N) retention there was an interaction (p<0.05) between diet type and phytase as the effects of phytase on N retention were more pronounced in the modified diets, with an increase from 0.512 to 0.561. These results demonstrate the positive effects of phytase on protein and energy utilisation, in addition to its established liberation of phytate-bound P and illustrate the feasibility of assigning nutrient replacement values to the feed enzyme for consideration in least-cost ration formulations. Further work is, however, required to define the most appropriate reductions in nutrient specifications in association with phytase supplementation.

Protein utilisation and turnover in lines of chickens selected for different aspects of body composition

1. Protein utilisation and turnover were measured in male chickens sampled from a line selected for high breast yield and a randombred control line (lines QL and CL, experiment 1) and in male chickens sampled from lines selected for either high or low abdominal fatness (lines FL and LL, experiment 2). In each experiment, 18 birds per line were given iso-energetic (12.9 MJ ME/kg) diets containing either 120 or 220 g CP/kg from 21 to 29 d (experiment 1) and 33 to 43 d (experiment 2). 2. Measurements were made of growth rate, food intake, body composition, excreta production and N-tau-methylhistidine excretion as a measure of myofibrillar protein breakdown, and fractional rates (%/d) of protein deposition, breakdown and synthesis were calculated. 3. In experiment 1, there were no significant differences between the line means for the fractional measures of protein turnover, but there was marked differential response in the two lines in the fractional rates of protein deposition, breakdown and synthesis, to increase in protein intake. The positive slope of the regressions of fractional (%/d) protein deposition and synthesis rates on protein intake (g/d/kg BW) were approximately 1.4- and 2.0-fold higher respectively in the QL than the CL line birds, and the negative slope of the regression of fractional breakdown rate on protein intake was approximately threefold greater in the CL than the QL line birds. 4. In experiment 2, fractional deposition rate was 6.2% lower, but fractional breakdown rate 9.4% higher in the LL than the FL birds, whilst there was essentially no difference in response of the FL and LL birds in the components of protein turnover to increase in protein intake. Line differences in deposition and breakdown rates were thus a reflection of the considerably higher (20%) food and hence protein intake in the FL than the LL birds. 5. The differential line responses in protein turnover in the two experiments suggest that selection for increased breast muscle yield and for reduced body fatness manipulate different physiological pathways in relation to protein turnover, but neither selection strategy results in an improvement in net protein utilisation at typical levels of protein intake by birds on commercial broiler diets, through a reduction in protein breakdown rate.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

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.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design: Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting: Investigational intensive care unit at a university hospital. Subjects: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mu g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results., In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in PaO2/FiO(2) ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Mechanisms underlying the diminished sensitivity to prolactin negative feedback during lactation: Reduced STAT5 signaling and up-regulation of cytokine-inducible SH2 domain-containing protein (CIS) expression in tuberoinfundibular dopaminergic neurons

Hyperprolactinaemia during lactation is a consequence of the sucking stimulus and in part due to reduced prolactin (PRL) negative feedback. To date, the mechanisms involved in this diminished sensitivity to PRL feedback are unknown but may involve changes in PRL signal transduction within tuberoinfundibular dopaminergic (TIDA) neurons. Therefore, we investigated signal transducers and activators of transcription (STAT) 5 signaling in the TIDA neurons of lactating rats. Dual-label confocal immunofluorescence studies were used to determine the intracellular distribution of STAT5 within TIDA neurons in the dorsomedial arcuate nucleus. In lactating rats with pups removed for 16 h, injection of ovine PRL significantly (P < 0.05) increased the STAT5 nuclear/cytoplasmic ratio compared with vehicle-treated mothers. In contrast, ovine PRL injection did not increase the STAT5 nuclear/cytoplasmic ratio in lactating mothers with pups, demonstrating that PRL signal transduction through STAT5 is reduced in TIDA neurons in the presence of pups. To investigate possible mechanisms involved in reduced PRL signaling, we examined the expression of suppressors of cytokine signaling (SOCS) proteins. Northern analysis on whole hypothalamus showed that CIS (cytokine-inducible SH2 domain-containing protein), but not SOCS1 or SOCS3, mRNA expression was significantly (P < 0.01) up-regulated in suckled lactating rats. Semiquantitative RT-PCR on arcuate nucleus micropunches also showed up-regulation of CIS transcripts. Immunofluorescence studies demonstrated that CIS is expressed in all TIDA neurons in the dorsomedial arcuate nucleus, and the intensity of CIS staining in these neurons is significantly (P < 0.05) increased in lactating rats with sucking pups. Together, these results support the hypothesis that loss of sensitivity to PRL-negative feedback during lactation is a result of increased CIS expression in TIDA neurons.