Searching for serum protein markers of equine squamous gastric disease using gel electrophoresis and mass spectrometry.

BACKGROUND: Equine squamous gastric disease (ESGD) is a very common disorder but an accurate and practical screening technique for detecting ESGD is currently lacking. OBJECTIVES: To identify serum protein markers to detect ESGD using electrophoresis and mass spectrometry. STUDY DESIGN: Proteomic analysis and bioinformatics. METHODS: ESGD was diagnosed using gastroscopy in 30 horses. Gastric ulceration was categorised into three groups: normal, mild/moderate and severe ESGD. Pooled sera from each group were compared using 1D electrophoresis and mass spectrometry. The candidate proteins for ESGD markers were selected based on their specifically high expression in nonglandular stomach, and their association with gastric ulceration using public gene and protein databases. RESULTS: The prevalence of ESGD in this study was 43% (with mild/moderate ESGD at 33% and severe ESGD at 10%). The proteomic study revealed that the identified serum protein markers for normal equine stomach were B4GALNT2 and XDH. The marker for mild/moderate EGSD was KRT10, while the marker for severe ESGD was KLK13. Furthermore, markers for both ulcer types were SLC4A7, PPARG, FCGBP, PKP1, ASPRV1 and KRT5-like proteins. MAIN LIMITATIONS: The functions of the identified proteins are not well characterised in horse. Proteomics is a tool for screening protein markers, but confirmation of putative protein markers with specific antibodies is required. CONCLUSIONS: In total, 10 serum proteins found in this study may be used as putative markers for ESGD. However, confirmation of candidate proteins with specific antibodies in a larger study cohort is necessary before it can be used in the veterinary clinic or on horse farms. The Summary is available in Portuguese - see Supporting Information.

Effects of bethanechol on canine urinary bladder smooth muscle function.

We studied whether the effects of bethanechol are mediated via a muscarinic receptor, the role of extracellular calcium on bladder contraction, and down-regulation of bladder contraction by bethanechol after activation with potassium chloride (KCl) and acetylcholine (Ach). Smooth muscle strips of normal urinary bladder were studied with standard methods to measure isometric force. Bethanechol caused a dose-dependent increase in bladder contraction. The potency of bethanechol is higher than Ach, as shown by higher peak active isometric stress (P(max)) and lower half-maximal contraction (ED(50)) (P< 0.01). The contractile responses to bethanechol were diminished in the presence of atropine, nifedipine and in calcium-free medium as shown by P(max) decreased by 58%, 87% and 65% and ED(50) increased by 314-, 24- and 16-fold, respectively. When bladder strips were stimulated with KCl and Ach, pre-treatment with bethanechol reduced the responses to KCl by 116-242% (P<0.05), while the contractile responses to Ach were unaltered. Thus, bethanechol induces bladder contraction via muscarinic receptor activation while both intracellular and extracellular calcium play a crucial role on bladder smooth muscle contraction. The mechanisms of down-regulation by bethanechol may be related to interference with calcium influx into the smooth muscle cells, rather than the desensitisation of muscarinic receptors or post-receptor steps of signal transduction following bethanechol binding to the receptor.

Influence of caecectomy and dietary protein concentration on apparent excreta amino acid digestibility in adult cockerels.

1. The influence of caecectomy and dietary crude protein content (50, 100, 150, 200 and 250 g/kg diet) on the apparent excreta amino acid digestibilities in adult cockerels fed on semi-purified diets containing soyabean meal (SBM) or cottonseed meal (CSM) as the sole source of protein was investigated. 2. Caecectomy had no influence on the apparent digestibilities of most amino acids in SBM, the exceptions being histidine, arginine and lysine, but lowered the apparent digestibility of amino acids in CSM. 3. The protein content in assay diets has a strong influence on the apparent amino acid digestibility values for poultry. Apparent values are lower when the dietary protein content is low. 4. The use of a single endogenous amino acid output value, generated by regression analysis or protein-free diets, for true digestibility adjustments across a wide range of protein intakes is questioned. In particular, such an application will penalise estimates of apparent digestibility when the dietary protein content is high.

Homoarginine influences voluntary feed intake, tissue basic amino acid concentrations and arginase activity in chickens.

Two experiments were conducted to investigate the factors responsible for the adverse effects of guanidinated proteins on feed intake in chickens. In Experiment 1, male broiler chicks were fed one of five purified diets containing casein or guanidinated casein (G-casein) as the sole source of protein (230 g crude protein/kg diet) from d 6 to 13 post-hatching. A casein-based diet containing 17.2 g lysine/kg, served as the control. In the experimental diets, casein was substituted by G-casein and lysine was added at 0, 5.6, 11.4 and 17.0 g/kg diet, respectively. Feed intake and weight gains of chicks fed the G-casein diet without added lysine were markedly depressed (P < 0.05), but this depression was largely overcome by additional lysine. The intake and gains of chicks fed the G-casein diet plus 17.0 g lysine/kg were lower (P < 0.05) than those fed the G-casein diet plus 11.4 g lysine/kg and this was associated with a higher plasma lysine:arginine ratio. Tissue analysis showed that homoarginine is distributed throughout body tissues following absorption. Brain lysine concentrations were lower (P < 0.05) in chicks fed diets containing G-casein without added lysine, but increased (P < 0.05) with supplemental lysine. In Experiment 2, the effect of homoarginine per se on feed intake was investigated in two short-term intake studies using 5-wk-old broiler chickens. Significant (P < 0.05) depressions in feed intake were observed within the first hour after oral administration of 400 mg homoarginine-HCl. The results suggest that both lysine deficiency and homoarginine per se were responsible for the adverse effects of guanidinated proteins on feed intake in chickens.

Secretion of homoarginine into the gut of chickens.

A technique, based on the homoarginine present in guanidinated proteins, has been used to distinguish between endogenous secretions and exogenous dietary amino acids in the ileal digesta of monogastric animals. This technique assumes that the ingested homoarginine is not recycled into the small intestine after absorption, but this assumption is yet to be experimentally validated in chickens. The secretion of homoarginine into the gut of broilers that were intravenously infused with 20 and 40 mmol/L homoarginine solutions was assessed. The plasma concentrations of homoarginine increased with increasing concentrations of homoarginine infused. However, only negligible levels of homoarginine (7.0 to 45.2 micrograms/g dry matter) were found in the digesta. Less than 0.01% of the intravenously infused homoarginine was recovered in the intestinal digesta, indicating that the secretion of homoarginine into the gut of chickens was insignificant.

Evaluation of homoarginine as a marker for the determination of endogenous amino acid concentrations in poultry excreta.

1. Endogenous amino acid losses in ileal digesta and excreta of adults cockerels fed on diets containing guanidinated forms of casein, soyabean meal and cottonseed meal were determined using homoarginine as a marker. 2. The ileal endogenous amino acid losses were markedly higher (P < 0.001) in birds given the cottonseed meal diet compared to those given the other two diets. The ileal endogenous protein was rich in aspartic acid, serine and glutamic acid. 3. Negative values were obtained for endogenous amino acid output in excreta. These aberrant values were caused by high concentrations of homoarginine in the excreta. A subsequent study with broiler chickens showed that the homoarginine in excreta was of urinary origin. 4. These results indicate that the homoarginine technique is not suitable for determining endogenous amino acid losses in excreta, but applicable when determinations are made in the terminal ileum.

Additivity of apparent and true ileal amino acid digestibilities in soybean meal, sunflower meal, and meat and bone meal for broilers.

An experiment was conducted to determine the additivity of apparent and true ileal amino acid digestibility values in soybean meal (SBM), sunflower meal (SFM), and meat and bone meal (MBM). A total of 63 individually caged 5-wk-old broilers were assigned to seven groups and given semi-purified diets containing SBM, SFM, MBM, and their combinations. True digestibilities were estimated by using the homoarginine in guanidinated proteins as the marker. Additivity was tested by comparing the differences between the observed digestibilities of ingredient combinations and the predicted values from measurements with individual ingredients. In general, for both apparent and true digestibilities, there were no significant differences (P > 0.05) between the observed and predicted values in ingredient combinations. The only exception was SBM + MBM combination, in which the observed values for apparent digestibilities of aspartic acid, serine, glutamic acid, isoleucine, and tyrosine were significantly higher (P < 0.05) than the predicted values. Overall, the present results indicate that amino acid digestibility values are additive and that digestible amino acid supply in a complete diet can be predicted from amino acid digestibilities of individual ingredients.

Non-starch polysaccharide-degrading enzymes increase the performance of broiler chickens fed wheat of low apparent metabolizable energy.

The effect of a commercial glycanase product (Avizyme TX) on the performance of 4-wk-old broiler chickens fed wheats with low and normal apparent metabolizable energy values was studied. Controls were fed a corn-based diet. Supplementation with the enzyme product significantly (P < 0.01) increased the apparent metabolizable energy of the low metabolizable energy wheat from 12.02 to 14.94 MJ/kg dry matter. The apparent metabolizable energy value of the normal wheat was increased from 14.52 to 14.83 MJ/kg dry matter; this was, however, not significant. Birds fed the low metabolizable energy wheat diet had significantly (P < 0.01) higher digesta viscosity and lower small intestinal starch and protein digestibilities than birds fed the normal wheat diet. Chickens fed the low metabolizable energy wheat tended to grow less than those fed the normal wheat diet. When the low metabolizable energy wheat+enzyme diet was fed, digesta viscosity was significantly (P < 0.01) lower (20.28 vs. 10.36 mPa.s), and small intestinal digestibility coefficient of starch was significantly (P < 0.01) greater (0.584 vs. 0.861) relative to values in birds fed the low metabolizable energy wheat diet alone. Although the protein digestibility coefficient also increased from 0.689 to 0.745, the difference was not significant. Weight gain and feed efficiency of birds fed the low metabolizable energy wheat+enzyme equaled those of controls. The enzyme product significantly (P < 0.01) increased the solubilization of non-starch polysaccharides within the gastrointestinal tract of birds fed both types of wheat diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of calcium absorptive and secretory capacities of segments of intact or functionally resected intestine during normo-, hypo-, and hyper-calcemia.

Absorptive and secretory capacities of six in situ intestinal loops of equal length were compared under the same calcium load and calcemic condition. The highest rate of calcium absorption was found in duodenum, colon, and proximal jejunum when loops were filled with 0.3 mM calcium, and in duodenum and proximal jejunum when filled with 10 mM luminal calcium. Secretory rates were in the following order: duodenum, jejunum, proximal jejunum, cecum, ileum, and proximal colon. Absorption of 0.3 mM calcium was decreased in all but the cecum and colon during hypercalcemia, and in duodenum, proximal jejunum, and colon during thyroparathyroidectomy-induced hypocalcemia. In contrast, calcium secretion was directly related to plasma calcium concentration and the length of the intestine. Functional resection of any part met with a compensatory increase in calcium absorption by the remaining segments, with the exception of the resection of the distal ileum with the large bowel. In conclusion, proximal small intestine exhibited the highest rate of absorption and secretion, but functional resection of this or any part did not affect the overall calcium absorption if luminal calcium was 10 mM. Moreover, enhanced secretion and reduced absorption during hypercalcemia were beneficial with respect to plasma calcium regulation.