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Este documento fue originado durante la “Primera Reunión del Núcleo de Estudio de Nuevas Técnicas de Mejoramiento Genético” del PROCISUR, realizada en Montevideo entre el 25 y 26 de agosto de 2017 y redactado, en versiones corregidas, entre esa fecha y el 16 de noviembre de 2018. Especialistas de los INIAs que integran el PROCISUR alientan el debate en torno a la ingeniería genética, a partir de los avances que se están alcanzando con la edición génica. Analizan el potencial de las tecnologías de modificación genética que tiene un consecuente impacto en el aumento de la variabilidad. La incidencia actual y futura en la generación de animales y plantas mejoradas, debido a su menor costo y mayor accesibilidad, presenta oportunidades en cultivos de reproducción agámica como la papa, el banano, la yuca, la caña de azúcar o la vid, entre otros. Reconocen que la utilización de esta herramienta puede modificar sustancialmente el esquema de los programas de mejoramiento, ya que permitiría realizar mejoras incrementales sobre genotipos establecidos y adaptados. EEA Balcarce Fil: Feingold, Sergio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Bonnecarrère, Victoria. Instituto Nacional de Investigación Agropecuaria (INIA); Uruguay Fil: Nepomuceno, Alexandre. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Brasil Fil: Hinrichsen, Patricio. Instituto de Investigaciones Agropecuarias (INIA); Chile. Fil: Cardozo Tellez, Lourdes Maria. Instituto Paraguayo de Tecnología Agrícola (IPTA); Paraguay. Fil: Molinari, Hugo. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Brasil Fil: Barba, Paola. Instituto de Investigaciones Agropecuarias (INIA); Chile Fil: Eyherabide, Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino; Argentina Fil: Ceretta, Sergio Eduardo. Instituto Nacional de Investigación Agropecuaria (INIA); Uruguay Fil: Dujack, Christian. Instituto Paraguayo de Tecnología Agrícola (IPTA); Paraguay.

In this study natural-based complex polyphenols, obtained through a smart synthetic approach, have been evaluated for their ability to inhibit the formation of A? oligomers, the most toxic species causing synaptic dysfunction, neuroinflammation, and neuronal death leading to the onset and progression of Alzheimer's disease. In vitro neurotoxicity tests on primary hippocampal neurons have been employed to select nontoxic candidates. Solution NMR and molecular docking studies have been performed to clarify the interaction mechanism of A? with the synthesized polyphenol derivatives, and highlight the sterical and chemical requirements important for their antiaggregating activity. NMR results indicated that the selected polyphenolic compounds target A? oligomeric species. Combined NMR and docking studies indicated that the A? central hydrophobic core, namely, the 17-31 region, is the main interaction site. The length of the peptidomimetic scaffold and the presence of a guaiacol moiety were identified as important requirements for the antiaggregating activity. In vivo experiments on an A? oligomer-induced acute mouse model highlighted that the most promising polyphenolic derivative (PP04) inhibits detrimental effects of A? oligomers on memory and glial cell activation. NMR kinetic studies showed that PP04 is endowed with the chemical features of true inhibitors, strongly affecting both the A? nucleation and growth rates, thus representing a promising candidate to be further developed into an effective drug against neurodegenerative diseases of the amyloid type.

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic BPera^ sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacteriummediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm2) than in the transgenic plants (

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a SS link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured ?1 rotameric states (t, g+ and g- for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand

Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polar residues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a K-d upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions.

summary of the identified exchange peaks observed to- gether with their chemical shifts is reported in Table 1. Their assignment was achieved assuming that each resonance of the native state is in exchange with the resonance of the correspond- ing proton in the molten globule state (e. g. the H2,6 resonance will interconvert directly with the corresponding H2’,6’ reso- nance, where the prime notation refers to protons in the molten globule state). A comparison between the chemical shifts of the molten globule species and the corresponding native resonances shows that, as previously observed by Shimizu et al. (1993), the former move towards the random coil values (Table l), with H4 of Trpl04 as the only exception. Exchange is observed for Trp26, Phe31, Tyr103, Trpl04 and Trpll8. The observation of these exchange peaks has also been reported by Baum et al. (1989) in a two-dimensional exchange spectrum of guinea pig a-lactalbumin and by Shimizu et al. (1993) for bovine a-lactal- bumin. The presence of exchange involving other residues, such as Tyrl8 and Phe53, could not be excluded from the ROESY, but could not be detected because of peak overlap. An estimate of the exchange rate was derived for the well separated resonances of Tyr103 H2,6/H2’,6’, Phe3 1 H2,6/H2’,6’ and Trpl04 H4/H4’ from the ratio between cross and diagonal peaks in the NOESY spectrum at 40 ms mixing time (Emst et al., 1987). Simplified equations were used based on the following assumptions: (a) the two sites are assumed equally populated; (b) both cross and spin lattice relaxation terms are equal for the two species; (c) an initial rate approximation is assumed. While the first two assumptions are rather arbitrary in a general case, the last one is expected to be reliable at a mixing time of 40 ms. Comparable interconversion rates are shown by Phe31 and Trpl04 (1.5 s-’ and 1.7

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine alpha-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.

The experimental assignment of most residues in a protein to the surface or interior is in principle possible without prior solution of a complete three-dimensional structure. The method described is based on nmr measurements that determine the amino acid composition of the surface of a protein [A. Petros, L. Mueller, and K.D. Kopple (1990) Biochemistry, Vol. 29, pp. 10041-10048; G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore (1992) Journal of Molecular Biology, Vol. 224, pp. 659-670]. If these measurements are carried out on several homologous proteins of known sequence, it is possible to combine the results to determine, in most cases, which positions in the sequence contain exposed residues.

Background Contradicting evidence has been presented in the literature concerning the effectiveness of empirical contact energies for fold recognition. Empirical contact energies are calculated on the basis of information available from selected protein structures, with respect to a defined reference state, according to the quasi-chemical approximation. Protein-solvent interactions are estimated from residue solvent accessibility. Results In the approach presented here, contact energies are derived from the potential of mean force theory, several definitions of contact are examined and their performance in fold recognition is evaluated on sets of decoy structures. The best definition of contact is tested, on a more realistic scenario, on all predictions including sidechains accepted in the CASP4 experiment. In 30 out of 35 cases the native structure is correctly recognized and best predictions are usually found among the 10 lowest energy predictions. Conclusion The definition of contact based on van der Waals radii of alpha carbon and side chain heavy atoms is seen to perform better than other definitions involving only alpha carbons, only beta carbons, all heavy atoms or only backbone atoms. An important prerequisite for the applicability of the approach is that the protein structure under study should not exhibit anomalous solvent accessibility, compared to soluble proteins whose structure is deposited in the Protein Data Bank. The combined evaluation of a solvent accessibility parameter and contact energy allows for an effective gross screening of predictive models.

A model based on the Poisson-Boltzmann equation has been used to model electrostatics in Anti-p24 (HIV-1) Fab-antigen association. The ionization state at different pH values has been simulated and the results have been used to estimate the stability at different pH values and to generate electrostatic potential maps at physiological ionic strength. The analysis of the electrostatic potential at the solvent-accessible surface shows that residues involved in binding are mostly found in the highest, but also in lowest potential regions. Brownian dynamics simulations have been used to estimate the enhancement of the association rate due to electrostatics which appears limited (approximately 2 at 150 mM ionic strength and approximately 3 at 15 mM ionic strength). A much more pronounced effect is observed upon increase of the charge of the diffusing particle. These results compare well with results obtained previously in similar studies on different systems and may serve to estimate the expected order of magnitude of electrostatic effects on association rates in antibody-antigen systems.