Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (Ka: 1560 ± 20 M-1). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested lycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design

Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible

Cultivo in vitro de Anaplasma marginale en líneas celulares endoteliales

Anaplasma marginale, intracellular bacteria causes bovine anaplasmosis, characterized by progressive anemia, fever, decrease in body weight and death occasionally. The use of A. marginale in vitro cell culture - derived has been suggested as an alternative for development new prevention tools and control strategies.Anaplasma marginale, bacteria intracelular obligada, es el agente causal de la anaplasmosis bovina, caracterizada por anemia progresiva, fiebre, pérdida de peso y ocasionalmente, la muerte. Como alternativa para el desarrollo de nuevas estrategias de prevención se plantea el uso de cultivos celulares para la obtención in vitro de A. marginale

Enthalpic nature of the CH/pi interaction involved in the recognition of carbohydrates by aromatic compounds, confirmed by a novel interplay of NMR, calorimetry, and theoretical calculations

10 páginas, 6 figuras, 2 tablas, 1 esquema -- PAGS nros. 18129-18138Specific interactions between molecules, including those produced by a given solute, and the surrounding solvent are essential to drive molecular recognition processes. A simple molecule such as benzene is capable of recognizing and differentiating among very similar entities, such as methyl 2,3,4,6-tetra-O-methyl-α-d-galactopyranoside (α-Me5Gal), methyl 2,3,4,6-tetra-O-methyl-β-d-galactopyranoside (β-Me5Gal), 1,2,3,4,6-penta-O-acetyl-β-d-galactopyranose (β-Ac5Gal), and methyl 2,3,4,6-tetra-O-methyl-α-d-mannopyranoside (α-Me5Man). In order to determine if these complexes are formed, the interaction energy between benzene and the different carbohydrates was determined, using Calvet microcalorimetry, as the enthalpy of solvation. These enthalpy values were −89.0 ± 2.0, −88.7 ± 5.5, −132.5 ± 6.2, and −78.8 ± 3.9 kJ mol−1 for the four complexes, respectively. Characterization of the different complexes was completed by establishing the molecular region where the interaction takes place using NMR. It was determined that β-Me5Gal is stabilized by the CH/π interaction produced by the nonpolar region of the carbohydrate on the α face. In contrast, α-Me5Man is not specifically solvated by benzene and does not present any stacking interaction. Although α-Me5Gal has a geometry similar to that of its epimer, the obtained NMR data seem to indicate that the axial methoxy group at the anomeric position increases the distance of the benzene molecules from the pyranose ring. Substitution of the methoxy groups by acetate moieties, as in β-Ac5Gal, precludes the approach of benzene to produce the CH/π interaction. In fact, the elevated stabilization energy of β-Ac5Gal is probably due to the interaction between benzene and the methyl groups of the acetyls. Therefore, methoxy and acetyl substituents have different effects on the protons of the pyranose ringThis work was supported by CONACYT through grants 49921-Q and 47679-Q, and by DGAPA-UNAM grant IN-209606. J.J.-B. Thanks “Ramón y Cajal” programme and MICINN (Spain), grant CTQ2006-10374-C02-01Peer reviewe

Protección contra babesiosis con una vacuna mixta de B.bovis y B.bigemina derivada de cultivo in vitro en una confrontación de campo. II Inmunización en una área endémica

The protection conferred by a combined in vitro culture-derived Babesia bovis and B. bigemina vaccine to susceptible Bos taurus bulls in a babesiosis endemic area was evaluated. Participating animals were over 18 months of age coming from a tick free area. Twenty animals were allocated at random into two groups of 10 animals each and taken to a tick endemic area. One week after arrival, 10 of them were vaccinated i.m. with an experimental Babesia spp. immunogen at a dose of 1 x 107 infected erythrocytes of each species. The 10 control animals were inoculated with uninfected red blood cells. All the animals were kept from the day of arrival until d 21 post vaccination in a pen free of Boophilus ticks and then released to a tick infested paddock where they remained until study',s completion. Twelve days after the start of the tick challenge, nine of the control animals presented poor physical condition, and also severe signs of babesiosis i.e. rectal temperature (RT) of more than 41 oC, a packed cell volume (PCV) drop of 46 % and presence of both species of Babesia in blood stained smears and had to be treated in order to avert their death. On the other hand, although the vaccinated animals presented a RT of 40.4 oC and a PCV drop of 40.7 %, only three animals showed poor physical condition and had to be treated on d 22 post introduction to the infected paddock. It can be concluded that a combined B. bovis and B. bigemina vaccine is able to confer a 70 % protection to bovines introduced to Babesia infected areasSe evaluó la protección de un inmunógeno bivalente de B. bovis y B. bigemina derivado de cultivo in vitro. Se utilizaron 20 toretes Bos taurus mayores de 18 meses provenientes de una área libre de garrapatas Boophilus, y se trasladaron a una zona endémica, formándose en forma aleatoria dos grupos de 10 animales. Una semana posterior a su llegada, 10 de los animales se vacunaron por vía intramuscular a una dosis de 1 x 107 eritrocitos infectados de cada especie del parásito, los 10 restantes (testigo) recibieron una dosis similar de eritrocitos no infectados. Los bovinos permanecieron desde el día de su llegada hasta el día 21 posvacunación, en un corral libre de garrapatas. Doce días posteriores a la introducción al potrero (PIP), nueve de los animales del grupo testigo presentaron muy mala condición corporal y severos signos de babesiosis, como temperatura rectal (TR) superior a los 41 oC, caída del volumen celular aglomerado (VCA) del 46 % y presencia de ambas especies del parásito en frotis sanguíneos, por lo que tuvieron que ser tratados para evitar su muerte. En contraste, y a pesar de presentar una TR de 40.4 ºC y un decremento en el VCA de 40.7 %, la condición corporal de los animales vacunados fue satisfactoria, con excepción de tres bovinos, que tuvieron que recibir tratamiento el día 22 PIP para evitar que muriesen. Se concluye que la vacuna mixta de B. bovis y B. bigemina produjo un 70 % de protección a bovinos recién introducidos en una zona endémica

Transmisión de cepas atenuadas de babesia bigemina y babesia bovis por garrapatas rhipicephalus (boophilus) microplus

To assess transmissibility of attenuated Babesia bigemina (BIS) and Babesia bovis (BOR) strains by Rhipicephalus microplus (R. microplus), 12 steers from a tick-free area were divided in four groups and infested at different intervals with R. microplus larvae. One animal from each group was inoculated with 1x108 infected erythrocytes (IE) of attenuated BIS, BOR, virulent B. bigemina, or B. bovis field strains.Para evaluar la transmisión por garrapatas Rhipicephalus microplus (R. microplus) de una clona atenuada de Babesia bovis (BOR) y una cepa atenuada de Babesia bigemina (BIS), doce bovinos se dividieron en cuatro grupos e infestados de forma escalonada con larvas de R. microplus libres de Babesia spp. Un becerro de cada grupo se inoculó con 1x108 eritrocitos infectados (EI) con BIS, BOR, y cepas virulentas de B. bigemina y B. bovis, respectivamente