Efecto sobre el rendimiento productivo y calidad de la canal de la inclusión de guisantes (Pisum sativum) y alberjón (Vicia narbonensis) en el pienso de lechones y cerdos de cebo

Las leguminosas grano presentan un perfil nutricional de gran interés para alimentación de ganado porcino, debido principalmente a su elevado contenido proteico. Sin embargo, la presencia de factores antinutritivos (FAN), que según el género difieren en calidad y cantidad, condiciona la absorción de la proteína, el nutriente más valorado. El objetivo de esta Tesis Doctoral ha sido el estudio del efecto de los principales FAN de guisante y alberjón sobre el rendimiento productivo, de canal y de piezas nobles, cuando sustituyen a la soja, parcial o totalmente, durante la fase estárter y el periodo de engorde de cerdos grasos. Con este motivo se llevaron a cabo 4 ensayos con machos castrados y la misma línea genética: híbrido Duroc x (Landrace x Large white). En el ensayo 1, se estudió la influencia de distintos niveles de inhibidores de proteasas (IP) en el pienso sobre la productividad de lechones durante la fase estárter (40 a 61 días de edad). Para ello, se utilizaron tres variedades de guisantes de invierno que contenían diferentes cantidades de IP, tanto de tripsina (IT) como de quimotripsina (IQ) [unidades de tripsina inhibida/mg (UTI), unidades de quimotripsina inhibida/mg (UQI): 9,87- 10,16, 5,75-8,62 y 12,55-15,75, para guisantes Cartouche, Iceberg y Luna, respectivamente] más elevadas que en la harina de soja 47 (HnaS) y en la soja extrusionada (SE) (UTI/mg - UQI/mg: 0,61-3,56 y 2,36-4,65, para HnaS y SE, respectivamente). El diseño experimental fue al azar, con cuatro tratamientos dietéticos que diferían en las fuentes proteicas y en la cantidad de IP, enfrentando un pienso control de soja a otros tres piensos con guisantes de invierno de las variedades indicadas, que sustituían parcialmente a la soja. Cada tratamiento se replicó cuatro veces, siendo la celda con 6 lechones la unidad experimental. Los animales que consumieron el pienso con guisante Cartouche tuvieron más ganancia media diaria (GMD) que el resto (P < 0,001) con el mismo consumo medio diario (CMD) e índice de conversión (IC). No hubo diferencias significativas entre los animales del pienso control y los que consumieron piensos con guisantes Iceberg y Luna. En el ensayo 2 la leguminosa objeto de estudio fue el alberjón y su FAN el dipéptido _Glutamyl-S-Ethenyl-Cysteine (GEC). El diseño y el periodo experimental fueron los mismos que en el ensayo 1, con cuatro dietas que variaban en el porcentaje de alberjones: 0%, 5%, 15% y 25%, y de GEC (1,54% del grano). Los lechones que consumieron el pienso con 5% tuvieron un CMD y GMD más elevado (P < 0,001), con el mismo IC que los animales pertenecientes al tratamiento 0%. Los índices productivos empeoraron significativamente y de manera progresiva al aumentar el porcentaje de alberjones (15 y 25%). Se obtuvieron ecuaciones de regresión con estructura polinomial que fueron significativas tanto para el nivel de alberjón como para la cantidad de GEC presente en el pienso. El ensayo 3 se efectuó durante el periodo de engorde, sustituyendo por completo la soja a partir de los 84 días de edad con las tres variedades de guisantes de invierno, observando el efecto sobre el rendimiento productivo, de canal y piezas nobles. El diseño, en bloques completos al azar, tuvo cuatro tratamientos según el guisante presente en el pienso y, por lo tanto, los niveles de IP: Control-soja, Cartouche, Iceberg y Luna, con 12 réplicas de 4 cerdos por tratamiento. De 84 a 108 días de edad los animales que consumieron los piensos Control-soja e Iceberg, tuvieron el mismo CMD y GMD, empeorando en los cerdos alimentados con Luna y Cartouche (P < 0,05). El IC fue igual en los tratamientos Control-soja e Iceberg, ocupando una posición intermedia en Cartouche y peor en los cerdos del pienso Luna (P < 0,001). De 109 a 127 días de edad la GMD y el IC fueron iguales, con un CMD más elevado en Control-soja e Iceberg que en los cerdos que consumieron Cartouche y Luna (P < 0,05). No hubo diferencias significativas durante el acabado (128 a 167 días de edad). Globalmente el CMD y GMD fueron más elevados en los cerdos que comieron los piensos Iceberg y Control-soja, empeorando por igual en los que comieron Cartouche y Luna (P < 0,05); el IC fue el mismo en todos los tratamientos. No se observaron diferencias en los datos relacionados con peso y rendimiento de canal y piezas nobles (jamón, paleta y chuletero), ni del contenido de grasa intramuscular en el lomo y proporción de ácidos grasos principales (C16:0, C18:0, C18:1n-9) en la grasa subcutánea. En el ensayo 4, realizado durante el periodo de engorde (60 a 171 días de edad), se valoró el efecto de dietas con distintos niveles de alberjones, y en consecuencia de su factor antinutritivo el dipéptido GEC, sobre el rendimiento productivo y la calidad de la canal y piezas nobles. El diseño fue en cuatro bloques completos al azar, con cuatro tratamientos según el porcentaje de inclusión de alberjón en el pienso: 0%, 5%, 15% y 25%, con 12 réplicas por tratamiento y cuatro cerdos en cada una de ellas. El tratamiento con 5% mejoró la GMD al final de la fase de cebo (152 días de vida) y, junto con el 0%, presentaron los resultados más favorables de peso e IC al final del ensayo (171 días de vida). Del mismo modo, el peso y rendimiento de canal fueron más elevados en los cerdos alimentados con los tratamientos 0% y 5% (P < 0,001). Piensos con el 15 y 25% de alberjones empeoraron los resultados productivos, así como el rendimiento y peso de canal. Sucedió lo mismo con el peso de las piezas nobles (jamón, paleta y chuletero), significativamente superior en 0% y 5% frente a 15% y 25%, siendo los cerdos que consumieron este último pienso los peores. Por el contrario el rendimiento de jamón y chuletero fue más elevado en los cerdos de los tratamientos 25% y 15% que en los que consumieron los piensos con 5% y 0% (P < 0,001); en el rendimiento de paletas se invirtieron los resultados, siendo mayores en los animales de los tratamientos 0% y 5% (P < 0,001). Se obtuvieron ecuaciones de regresión polinomial, para estimar las cantidades de inclusión de alberjones y de GEC más favorables desde el punto de vista productivo, así como los contrastes ortogonales entre los distintos tratamientos. ABSTRACT The grain legumes have a nutritional profile of great interest to feed pigs, mainly due to high protein content. However, the presence of antinutritional factors (ANF), which differ in quality and quantity according to gender, hinder the absorption of the protein, the most valuable nutrient. The aim of this thesis was to study the effect of the main ANF of pea and narbon vetch (NV) on productive performance, of the carcass and main lean cuts, when replacing soybean, partially or totally, during the starter phase and the fattening period of heavy pigs. For this reason were carried four trials with barrows and the same genetic line: Duroc hybrid x (Landrace x Large white). In trial 1, was studied the influence of different levels of protease inhibitors (PI) in the diet over productivity of piglets during the starter phase (40-61 days of age). For this, were used three varieties of winter peas containing different amounts of PI, both trypsin (TI) and chymotrypsin (CI) [inhibited units/mg trypsin (TIU), inhibited units/mg chymotrypsin (CIU): 9.87 - 10.16, 5.75 - 8.62 and 12.55 - 15.75, for peas Cartouche, Iceberg and Luna, respectively] higher than in soybean meal 47 (SBM) and soybeans extruded (SBE) (TIU/mg - CIU/mg: 0.61 - 3.56 and 2.36 - 4.65 for SBM and SBE, respectively). The design was randomized with four dietary treatments differing in protein sources and the amount of PI, with a control diet of soybean and three with different varieties of winter peas: Cartouche, Iceberg and Luna, which partially replace soybean. Each treatment was replicated four times, being the pen with 6 piglets the experimental unit. Pigs that ate the feed with pea Cartouche had better growth (ADG) than the rest (P < 0.001), with the same average daily feed intake (ADFI) and feed conversion ratio (FCR). There were no significant differences between piglets fed with control diet and those fed Iceberg and Luna diets. In trial 2 the legume under study was the NV and your ANF the dipeptide _Glutamyl FAN-S-Ethenyl-Cysteine (GEC). The experimental period and the design were the same as in trial 1, with four diets with different percentage of NV: 0%, 5%, 15% and 25%, and from GEC (1.52% of the grain). The piglets that consumed the feed containing 5% had higher ADG and ADFI (P < 0.05), with the same FCR that pigs belonging to the 0% treatment. Production rates worsened progressively with increasing percentage of NV (15 and 25%). Were obtained regression equations with polynomial structure that were significant for NV percentage and amount of GEC present in the feed. The test 3 was carried out during the fattening period, completely replace soy from 84 days of age with three varieties of winter peas, observing the effect on the yield, carcass and main lean cuts. The design, randomized complete blocks, had four treatments with different levels of PI: Control-soy, Cartouche, Iceberg and Luna, with 12 replicates of 4 pigs per treatment. From 84 to 108 days of age the pigs fed with Control-soy and Iceberg feed, had the same ADFI and ADG, worsening in pigs fed with Luna and Cartouche (P < 0.05). The FCR was similar in diets Control-soy and Iceberg, occupying an intermediate position in Cartouche and worse in pigs fed with Luna (P < 0.001). From 109-127 days of age the ADG and FCR were equal, with higher ADFI in pigs fed with Control-soy and Iceberg, regarding pigs fed with Cartouche and Luna (P < 0.05). There was no difference in the finishing phase (128-167 days of age). In global period, the ADFI and ADG were higher in pigs that ate Control-soy and Iceberg, and worse in those who ate Cartouche and Luna. The FCR was the same in all treatments. No significant differences were observed in the data related to weight and carcass yield, main lean cuts (ham, shoulder and loin chop) and intramuscular fat loin content and major fatty acids proportion (C16:0, C18:0, C18:1n-9) of subcutaneous fat. In experiment 4, made during the fattening period (60-171 days of age), was assessed the effect of diets with different levels of NV, and consequently of GEC, in the performance and quality of carcass and main lean cuts. There was a completely randomized design with four dietary treatments differing in percentage of NV: 0%, 5%, 15% and 25%, with 12 replicates per treatment and four pigs each. Treatment with 5% improved the ADG at the end of the fattening phase (152 days of age) and, together with 0%, showed the most favorable body weight and FCR at the end of the trial (171 days of age). Similarly, the weight and performance of carcass were higher for pigs fed with diets 0% and 5% (P < 0.05). Diets with 15 and 25% worsened the productive and carcass results. The weight of the main lean cuts (ham, shoulder and loin chop) was significantly higher in 0% and 5% vs 15% and 25%.The diet 25% was the worst of all. By contrast the performance of ham and loin chop was higher in pigs fed with diets 25% and 15%, that those who ate diets with 5% and 0% (P < 0.001); the results of shoulder performance were reversed, being greater in pigs feed with diets 0% and 5% (P < 0.001). Polynomial regression equations were obtained to estimate the percentage of NV and GEC more favorable from the point of view of production, and orthogonal contrasts between treatments.

Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/− or Cat D−/− antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/− splenocytes, as it did in Cat D−/− cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Expressed protein ligation: A general method for protein engineering

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Role of calpain in skeletal-muscle protein degradation

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl β-d-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

Single prenyl-binding site on protein prenyl transferases

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.