INCLUSIÓN DE HARINAS DE FOLLAJES ARBÓREOS Y ARBUSTIVOS TROPICALES (Morus alba, Erythrina poeppigiana, Tithonia diversifolia E Hibiscus rosa-sinensis) EN LA ALIMENTACIÓN DE CUYES (Cavia porcellus Linnaeus) / LEAF MEALS AND TROPICAL SHRUBBY FOLIAGE (Morus alba, Erythrina poeppigiana, Tithonia diversifolia AND Hibiscus rosa-sinensis) IN FEEDING GUINEA PIGS (Cavia porcellus Linnaeus)

La investigación se ejecutó en la Finca Experimental "La María" propiedad de la Universidad Técnica Estatal de Quevedo (UTEQ) localizada en el km 7¹/2 de la vía Quevedo-Mocache; Provincia de Los Ríos cuya ubicación geográfica de 1º 6' 23" de latitud sur y 79º 29' 12" de longitud oeste y a una altura de 73 m.s.n.m. El objetivo principal fue evaluar el comportamiento productivo de cuyes con la inclusión del 20% de harinas derivadas de follajes arbustivos y arbóreos tropicales. Se utilizaron 40 cuyes macho de 30 días de edad. Se empleó un diseño completamente al azar con cinco tratamientos, cuatro réplicas y la unidad experimental estuvo conformado por dos cuyes. Para determinar las diferencias entre medias de tratamientos se aplicó la prueba de Tukey (P≤0.05). Se evaluaron cinco dietas-tratamientos: (T0) dieta 100% balanceado, (T1) 80% dieta y 20% harina de Morus alba, (T2) 80% dieta y 20% harina de Erythrina poeppigiana, (T3) 80% dieta y 20% harina de Tithonia diversifolia, (T4) 80% dieta y 20% harina de Hibiscus rosa-sinensis. Las variables bajo estudio fueron: consumo de alimento de balanceado en materia seca (CABMS, g), ganancia de peso (GP, g), índice de conversión alimenticia (ICA) y rendimiento en canal (RC, %). La rentabilidad de los tratamientos se determinó a través de la relación beneficio-costo (R b/c). Los mayores (P<0.01) CABMS, GP-ICA y RC-Rentabilidad, la registraron los tratamientos: T0 (48.34 g MS animal-1 d-1), T1 (8.80 g animal-1 d-1 y 5.04) y el T3 (77.67% y 26.20%), respectivamente.

The research was carried out at the Experimental farm "La María" property of the State Technical University of Quevedo located at km 7¹/2 in road Quevedo-Mocache; Los Ríos province, with a geographical location of 1° 6' 23" south latitude and 79º 29' 12" west longitude, at 73 meters altitude. The aim was to evaluate the productive effect of leaf meal and tropical shrubs with inclusion 20%. Were used 40 male guinea pigs of 30 days age and a completely randomized design with five treatments and four repetitions, two male guinea pigs was used to study. A 56-days experiment was conducted, and was applied the Tukey test (P≤0.05) to determine differences. Five treatments in diets were evaluated: (T0) 100% balanced diet; (T1) 80% and 20% leaf meal Morus alba flour (T2); 80% diet and 20% Erythrina poeppigiana flour; (T3) 80% diet and 20% Tithonia diversifolia flour; (T4) 80% diet and 20% Hibiscus rosa-sinensis flour. The following variables were used: Balanced feed consumption in dry matter (CABMS, g), weight obtained (GP, g), alimentary conversion index (ICA), performance distribution channel (RC, %). The profitability of the treatments was determined using the benefit-cost ratio (R b/c) ratio. The higher (P<0.01) CABMS, GP-ICA, RC- yield, assigned treatments: T0 (48.34 g DM animal-1 d-1), T1 (8.80 g animal-1 d-1 and 5.04) and T3 (77.67 % and 26.20%), respectively.

The product of the MLD gene is a member of the membrane fatty acid desaturase family: overexpression of MLD inhibits EGF receptor biosynthesis.

Membrane fatty acid desaturases are responsible for inserting double bonds into specific positions in fatty acids. We have cloned a new member of the human membrane fatty acid (lipid) desaturase gene family, MLD. The derived amino acid sequence of MLD contains three consensus motifs, HX3H, HX2HH, and HX2HHXFP, that are characteristic of a group of membrane fatty acid desaturases. MLD is predicted to be a multiple membrane-spanning protein and is found to be extractable from particulate fractions with detergent but not salt or urea. MLD is widely expressed in human tissues and is localized to the endoplasmic reticulum. Cotransfection of MLD with the epidermal growth factor (EGF) receptor resulted in decreased expression of the receptor but did not affect platelet-derived growth factor receptor expression. MLD overexpression inhibited biosynthesis of the EGF receptor, suggesting a possible role of a fatty acid desaturase in regulating biosynthetic processing of the EGF receptor.

Enhanced degradation of EGF receptors by a sorting nexin, SNX1.

The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery. A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes.

Analysis of substrate recognition determinants in a synthetic peptide containing the Tyr 1173 autophosphorylation site of the epidermal growth factor receptor.

The epidermal growth factor receptor contains five autophosphorylation sites in its C-terminal region. Synthetic peptides based on the major autophosphorylation site at Tyr 1173 were tested as substrates of the intracellular domain of the epidermal growth factor receptor. A peptide containing acidic residues N-terminal to the substrate Tyr as well as the Tyr-Met-Xaa-Met motif of the insulin receptor substrate 1 had a Km value of 15 microM, the lowest value for a synthetic peptide reported to date. Another important residue contributing to substrate binding is the Tyr itself, or more specifically, the hydroxyl group of the Tyr. Substituting Phe for Tyr results in a peptide that is ineffective as an inhibitor of kinase phosphorylation. However, substitution of a Ser residue does not restore a functional substrate, indicating specificity for the Tyr hydroxyl. Secondary structure algorithms predicted that the peptide substrate based on the native sequence at Tyr 1173 would have a propensity to adopt a helical conformation in solution. Circular dichroism spectroscopy confirmed this prediction. The secondary structure of the peptide substrate is significant in its consistency with the idea that secondary structure is an important determinant in substrate recognition by protein tyrosine kinases.

The intracellular tyrosine kinase domain of the epidermal growth factor receptor undergoes a conformational change upon autophosphorylation.

The intracellular portion of the epidermal growth factor receptor consists of a tyrosine kinase domain of approximately 290 amino acids and a COOH-terminal regulatory domain of approximately 230 amino acids that contains five sites of autophosphorylation. The effect of autophosphorylation on the conformation of the intracellular domain has been analyzed using gel filtration. Both phosphorylated and dephosphorylated forms of the intracellular domain exist as monomers and as dimers and appear to have an extended conformation. The Stokes' radii of phosphorylated monomers and dimers were larger than those of the dephosphorylated forms, indicating that the dephosphorylated form is more compact. These results indicate that a significant conformational change occurs in the intracellular portion of the epidermal growth factor receptor upon tyrosine autophosphorylation.

Expression and purification of the epidermal growth factor receptor extracellular domain utilizing a polycistronic expression system.

High level expression of the epidermal growth factor receptor ectodomain (EGFR-ED) has been achieved using a polycistronic expression system. The expression vector was designed such that EGFR-ED was at the 5' end of a tricistron followed by luciferase and dihydrofolate reductase (dhfr). Following transfection into Chinese hamster ovary cells, clones were isolated under selective conditions for dhfr expression and monitored for luciferase activity and EGFR-ED expression using immunofluorescence microscopy. A 100-kDa protein corresponding to EGFR-ED was efficiently secreted from expressing cells. Two purification schemes were used to obtain protein at least 95% pure. Glutaraldehyde crosslinking was used to show that EGFR-ED specifically binds EGF and TGF alpha and that the affinity for EGF is 5.5 x 10(-7) M.

Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition.

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.

Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II.

Mammalian cells contain two subspecies of RNA polymerase II, designated IIO and IIA. The objectives of these studies were to determine the structural relationship between these subspecies and to determine the functional significance of these differences. Subunits IIo and IIa were purified from calf thymus, and the effect of alkaline phosphatase treatment on electrophoretic mobility and immunochemical reactivity was examined. The removal of phosphate converts subunit IIo to a form indistinguishable from that of subunit IIa. These results indicate that subunit IIo is produced by multisite phosphorylation of subunit IIa. The distribution of phosphate within subunit IIo was determined by CNBr cleavage of in vivo labeled HeLa cell RNA polymerase II. 32P-Labeled subunit IIo was purified by immunoprecipitation and cleaved with CNBr, and the resultant peptides were analyzed. The quantitative recovery of 32P in the C-terminal peptide establishes that this domain is the primary site of phosphorylation. In an effort to assess the level of phosphorylation of the transcriptionally active form of RNA polymerase II in HeLa nuclei, transcription was carried out in the presence of 4-thiouracil triphosphate and the nascent labeled transcript cross-linked to RNA polymerase. Specific photoaffinity labeling of subunit IIo was observed. Alkaline phosphatase treatment results in an increase in the mobility of photoaffinity labeled subunit IIo to approach that of subunit IIa. These results indicate that subunit IIo is a component of transcriptionally active RNA polymerase II.

A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II.

Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites.