Use of phytase and citric acid supplementation on growth performance and nutrient digestibility of Cirrhinus mrigala fingerlings fed on canola meal based diet / Uso de suplementação de fitase e ácido cítrico no desempenho do crescimento e digestibilidade de nutrientes de dedos Cirrhinus mrigala alimentados em dieta à base de refeição de canola

Fishmeal; being a limited and costly feed ingredient is continuously been substituted with locally available plant proteins. However, the occurrence of anti-nutritional factors in plant meal suppresses its potential to be fully replaced. Therefore, in this study we aimed to study the synergistic effects of dietary additives like citric acid and phytase enzyme supplementation on growth performance and nutrient digestibility of Cirrhinus mrigala fingerlings. Canola meal (CM) was used as a test ingredient to replace fishmeal (FM) as; 0%, 25%, 50% and 75%. These four diets were further supplemented by varying levels of phytase (0 and 750 FTU kg-1) and citric acid (0% and 2.5%) to formulate total sixteen test diets as T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15 and T16. Each treatment contained three replicates; applied to fish groups having 15 fingerlings each; following 3×3 factorial arrangement. 1% of chromic oxide was added as an inert marker. Maximum weight gain% (288%) and the lowest value of FCR (1.07) were recorded when fish was fed on diet T12 as compared to fish fed control diet (T1). Similarly, optimum nutrient digestibility values such as crude protein (77%), crude fat (84%) and gross energy (70%) were noted on same level. It was concluded that 50% canola meal can optimally replace fishmeal when supplemented with phytase and citric acid at the levels of 750 FTU kg-¹ and 2.5%, respectively.

A farinha de peixe, por ser um ingrediente alimentar limitado e caro, é continuamente substituída por proteínas vegetais disponíveis localmente. No entanto, a ocorrência de fatores antinutricionais na farinha de plantas suprime seu potencial de ser totalmente substituída. Portanto, neste estudo objetivamos estudar os efeitos sinérgicos de aditivos dietéticos como ácido cítrico e suplementação com enzima fitase sobre o desempenho de crescimento e digestibilidade de nutrientes de alevinos de Cirrhinus mrigala. A farinha de canola (CM) foi usada como ingrediente de teste para substituir a farinha de peixe (FM) como: 0%, 25%, 50% e 75%. Essas quatro dietas foram suplementadas por níveis variados de fitase (0 e 750 FTU kg-1) e ácido cítrico (0% e 2,5%) para formular um total de 16 dietas de teste como T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15 e T16. Cada tratamento continha três repetições; aplicado a grupos de peixes com 15 alevinos cada; seguindo o arranjo fatorial 3 × 3. 1% de óxido crômico foi adicionado como um marcador inerte. % de ganho de peso máximo (288%) e o valor mais baixo de FCR (1,07) foram registrados quando os peixes foram alimentados com dieta T12 em comparação com peixes alimentados com dieta controle (T1). Da mesma forma, valores ótimos de digestibilidade de nutrientes, como proteína bruta (77%), gordura bruta (84%) e energia bruta (70%) foram anotados no mesmo nível. Concluiu-se que 50% da farinha de canola pode substituir de forma ideal a farinha de peixe quando suplementada com fitase e ácido cítrico nos níveis de 750 FTU kg-¹ e 2,5%, respectivamente.

Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors.

This review focuses on phytase functionality in the digestive tract of farmed non-ruminant animals and the factors influencing in vivo phytase enzyme activity. In pigs, feed phytase is mainly active in the stomach and upper part of the small intestine, and added phytase activity is not recovered in the ileum. In poultry, feed phytase activities are mainly found in the upper part of the digestive tract, including the crop, proventriculus and gizzard. For fish with a stomach, phytase activities are mainly in the stomach. Many factors can influence the efficiency of feed phytase in the gastrointestinal tract, and they can be divided into three main groups: (i) phytase related; (ii) dietary related and (iii) animal related. Phytase-related factors include type of phytase (e.g. 3- or 6-phytase; bacterial or fungal phytase origin), the pH optimum and the resistance of phytase to endogenous protease. Dietary-related factors are mainly associated with dietary phytate content, feed ingredient composition and feed processing, and total P, Ca and Na content. Animal-related factors include species, gender and age of animals. To eliminate the antinutritional effects of phytate (IP6), it needs to be hydrolyzed as quickly as possible by phytase in the upper part of the digestive tract. A phytase that works over a wide range of pH values and is active in the stomach and upper intestine (along with several other characteristics and in addition to being refractory to endogenous enzymes) would be ideal.

[Phosphate containing enemas: one undervalued risky practice? The management of an adverse event]. / Gli enteroclismi a base di fosfati: una pratica rischiosa sottovalutata? La gestione di un evento avverso.

INTRODUCTION: The phosphate-containing enemas are widely used, both to manage constipation and as a preparation for endoscopic procedures and surgery in adults and children. Many studies report that the use of these laxatives can be dangerous. OBJECTIVE: To identify possible prevention strategies starting from a severe adverse reaction due to repeated administrations of phosphate enemas. METHODS: A working group was started, the literature was reviewed and recommendations for an appropriate use of enemas were discussed and implemented, to improve patients' safety. RESULTS: Phosphate-containing enemas were replaced with 125 ml water enemas; recommendations were spread to strongly limit the use of phosphate containing enemas and the use of laxative in the first and second semester of 2012, were confronted showing a change in habits and a reduction in the use of phosphate containing enemas. CONCLUSIONS: The implementation of several strategies, originated from an adverse event, succeeded in modifying the use of laxatives and phosphate-containing enemas.

Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.

The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.

Estado nutricional, consumo de lácteos y niveles séricos de calcio, fósforo y fosfatasas alcalinas en escolares de mérida / Nutritional status, consumption of dairy products and levels sericos of calcium, phosphorus, and alkaline phosphatases in schoolchildren of mérida

Se realizó una investigación de Campo de Tipo Descriptiva Correlacional y de corte transversal para determinar el estado nutricional, consumo de lácteos y niveles séricos de calcio, fósforo, y fosfatasas alcalinas en escolares del 1er, 3er y 5to grado de la U.E "Rafael Antonio González" de la comunidad de Mesa Bolívar en el año 2007. La población estuvo conformada por la matricula escolar de 171 estudiantes. Se determinó la muestra con el método estratificado aleatorio simple, obteniéndose 47% de la matricula escolar, correspondiendo 80 niños distribuidos por grado: 21 niños en 1ero, 28 en 3ero y 31 en 5to, en edades comprendidas entre 6 a 12 años. Se determinó la cantidad y la frecuencia de consumo de productos lácteos para lo cual, se diseñó un cuestionario "ad hoc" contentivo de 10 ítems relacionados con la frecuencia de consumo, cantidad y tipo de lácteos. Se realizó evaluación nutricional a través de la Combinación de Indicadores (Peso para la Talla y Talla para la Edad) utilizando las tablas de Evaluación de la Organización Mundial de la Salud. Se determinaron los valores séricos de calcio, fósforo y fosfatasas alcalinas. Los escolares presentan 32,6% de malnutrición; tanto los niños (6-10 años y 11-12 años) como las niñas (8-12 años) presentaron un porcentaje de adecuación diario de calcio bajo (77,16%, 28,57% y 38,96%) respectivamente y 60% tienen hipocalcemia. Existe significancia estadística entre los niveles séricos de calcio y fósforo con el consumo diario promedio de calcio (p 0,05 y p 0,04). No hubo relación estadísticamente significativa entre el consumo de productos lácteos y el estado nutricional de los escolares. El estado nutricional de los escolares no depende del consumo diario de productos lácteos, sin embargo, dicho consumo si afecta los niveles séricos de calcio y fósforo(AU)

A cross-sectional descriptive correlational field research was conducted in order to determine the nutritional status, consumption of milk and serum levels of calcium, phosphorus, and alkaline phosphatase in students of 1st, 3rd and 5th grades of the "Rafael Antonio Gonzalez "school in Mesa Bolívar in 2007. The population consisted of 171 students. We determined the sample with a simple random stratified method, yielding 47% of school enrollment, corresponding to 80 children distributed by grade: 21 children in 1st, 28 in 3rd, 31 in 5th, aged 6 to 12 years old. The amount and frequency of consumption of dairy products, with an "ad hoc" questionnaire designed containing 10 items related to the frequency of consumption, quantity and type of dairy product. Nutritional assessment was carried out by means of the combination of indicators (weight for height and height for age) using the tables of evaluation of the World Health Organization. Values were determined in serum calcium, phosphorus and alkaline phosphatase. The students had 32,6% of malnutrition, both boys (6-10 years and 11-12 years) and girls (8-12 years) had an adequate percentage of low calcium daily intake(77.16%, 28. 57% and 38.96%, respectively) and 60% had hypocalcemia. There is statistical significance between serum calcium and phosphorus with an average daily intake of calcium (p 0.05 and p 0.04). There was no statistically significant relationship between dairy products consumption and nutritional status of schoolchildren. The nutritional status of schoolchildren does not depend on daily consumption of dairy products, however, that consumption does affect serum calcium and phosphorus(AU)

Cyclosporine exposure and calcineurin phosphatase activity in living-donor liver transplant patients: twice daily vs. once daily dosing.

We have compared the pharmacokinetics and pharmacodynamics of cyclosporine between once- and twice-daily dosing regimens in de novo patients of living-donor liver transplantation (LDLT). A total of 14 patients were enrolled in this study, who had received cyclosporine microemulsion (Neoral) twice a day (BID, n = 5) or once daily in the morning (QD, n = 9) after transplantation. On postoperative day (POD) 6, the QD regimen significantly increased cyclosporine exposure; the blood concentration at 2 hours postdose (C2) and area under the concentration-time curve (AUC) for 4 hours (AUC(0-4)), compared with the BID regimen. Moreover, the area under the calcineurin (CaN) activity in peripheral blood mononuclear cells time-curve (AUA) for 12 hours (AUA(0-12)) and 24 hours (AUA(0-24)) were decreased by approximately 42 and 25% with the QD regimen relative to the BID regimen, respectively. The C2 level was significantly correlated with the AUC(0-4) (r2 = 0.95), which was negatively related to the AUA(0-12) with a large interindividual variability (r(2) = 0.59). However, a significant correlation was found between the AUA(0-12) or AUA(0-24) and CaN activity at trough time points. According to a maximum inhibitory effect attributable to the drug (E(max)) model, the mean estimates of E(max) and the C(b) value that gives a half-maximal effect (EC50) for CaN inhibition were not significantly different between the 2 groups, respectively. These findings suggest that a once daily morning administration of cyclosporine may improve oral absorption and help to provide an effective CaN inhibition early after LDLT. Furthermore, CaN activity at trough time points would be a single surrogate predictor for the overall CaN activity throughout dosing intervals following cyclosporine administration.

A comparison of two sources of phytase in liquid and dry forms in broilers.

Research with corn-soybean meal diets was conducted to compare phytase sources in commercial broilers. A Ca to nonphytate P (nPP) ratio of 2.5:1 was maintained in all diets. Experiments 1 and 2 were conducted from d 4 to 13 (experiment 1) or d 9 to 23 post-hatching (experiment 2) in batteries. The 10 treatments used in both experiment were: Diets 1 to 4 = 0.20, 0.25, 0.30, or 0.35% nPP; Diets 5 to 7 = diet 1 plus 100, 200, or 300 phytase units/kg of diet from Natuphos (NAT); and Diets 8 to 10 = diet 1 plus 100, 200, or 300 phytase units/ kg of diet from Ronozyme (RON). As nPP levels increased, daily gain (ADG), feed intake (ADFI), gain:feed, and toe and tibia ash percentage were linearly increased (P < 0.06) in experiments 1 and 2. Incremental addition of phytase, regardless of source, linearly increased (P < 0.07) ADG and ADFI in experiment 1. Broilers fed NAT had higher (P < 0.07) toe ash percentage in experiment 1 and 2, and higher (P < 0.02) ADG and ADFI in experiment 2 than those fed RON. In experiment 3, 3,360 broilers were used to test 2 dry phytase products from 0 to 41 d posthatching in a 3-phase feeding program. The treatments were: Diet 1) adequate Ca and nPP; Diet 2) nPP reduced by 0.20%; Diets 3 to 5 = diet 2 plus 300, 500, or 750 phytase units/kg of diet from NAT; and Diets 6 to 8 = Diet 2 plus 300, 500, or 750 phytase units/kg of diet from RON. Broilers fed the adequate Ca and nPP diet had improved ADG and ADFI overall, and tibia ash weight and percentage in all growth phases (P < 0.03) compared with those fed the reduced Ca and nPP diets. Overall, ADG and ADFI were linearly increased (P < 0.05) by incremental phytase addition in experiment 3. Both NAT and RON produce similar growth and bone ash traits in commercial broilers.

Microinjection strategies for the study of mitogenic signaling in mammalian cells.

First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture. In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner. A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts. These strategies include: 1. Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain. 2. Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities. 3. Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules. In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response. The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4). For in vivo studies, the c-fos promoter offers several unique advantages. Primarily, it is easy to manipulate. In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours. Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5). Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress. In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression. However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation). As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off.

Phosphotriesterase decreases paraoxon toxicity in mice.

The effect of phosphotriesterase (PTE) on the ip toxicity of paraoxon was studied in mice. The PTE preparation (0.1 ml; paraoxon-hydrolyzing activity, 1.5 mumol/min) was given iv. Cholinesterase activities were measured 2 hr after paraoxon administration. The PTE treatment, given 10 min before paraoxon, did not protect serum cholinesterase (ChE) against the inhibiting effect of paraoxon, but it clearly prevented the decrease of the brain ChE activity. In PTE-nontreated animals ChE was reduced by 60% at the paraoxon dose of 0.5 mg/kg, whereas in PTE-treated mice a significant reduction was not seen until a paraoxon dose of 2.0 mg/kg. The iv injection of PTE did prevent the decrease in brain ChE activity by paraoxon, when it was administered before or immediately after the paraoxon. PTE, injected 15 min after paraoxon, resulted in a minor protection in the brain ChE activities. The iv injection of PTE increased the serum paraoxon-hydrolyzing activity up to 5.1-fold. When the same amounts of PTE were administered ip, im, or sc, the increases in the hydrolyzing activities were 4.7-, 2.5-, and 1.8-fold, respectively. The activities returned to the normal level within 24 hr after the PTE. The elimination half-life of the activity of PTE administered iv was approximately 5.5 hr. In conclusion, PTE substantially prevents the toxicity of paraoxon in mice by hydrolyzing paraoxon in circulation.

Microinjection of acylphosphatase blocks Xenopus laevis oocytes maturation induced by ras-p21.

Ras proteins induce germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. The mechanism of action is still unresolved, although several hypotheses have been proposed. Acylphosphatase is a cytosolic enzyme that specifically catalyses the hydrolysis of the carboxylphosphate bond of acylphosphate for the removal of acylphosphate residues of various membrane pumps. A direct effect of acylphosphatase on the regulation of ionic balance of a cell by interaction with ionic membrane pumps has been proposed. We have analyzed the effect of microinjecting acylphosphatase, by itself or along with ras-p21 proteins or progesterone, into oocytes. The enzyme alone is unable to induce GVBD, but increases oocyte maturation induced by progesterone. By contrast, acylphosphatase blocked GVBD induced by microinjection of oncogenic ras-p21. These data suggest that acylphosphatase acts synergistically or antagonistically with factors involved in proliferating signals by altering the intracellular ionic conditions of the cell, conforming the hypothesis that the intracellular ionic condition of the cell is important in the induction of proliferating signals, and that its perturbation may have a serious effect on signal transduction.

Protection against tabun toxicity in mice by prophylaxis with an enzyme hydrolyzing organophosphate esters.

We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.