Sustratos electrohilados de poli(hidroxibutirato-co-hidroxihexanoato) con glicósidos para reparación de la lesión medular / Glycosides-bearing electrospun poly(hydroxybutyrate-co-hydroxyhexanoate) substrates for the repair of injured spinal cord

Objetivo: Obtención mediante electrohilado de fibras micro- y submicrométricas de poliésteres funcionalizadas con glicósidos que constituyen elementos estructurales de proteoglicanos, para su uso en la reparación del tejido medular. Material y métodos: Las fibras se prepararon a partir de disoluciones de poli(3-hidroxibutirato-co-3-hidroxihexanoato) con glicósidos sintéticos mediante electrohilado variando sistemáticamente las condiciones del proceso. La morfología de las fibras fue analizada mediante microscopía electrónica de barrido. Asimismo, se evaluó la estabilidad de la interacción entre el glicósido y la fibra en medio acuoso, y su toxicidad en cultivos de células neurales. Resultados: La morfología de las fibras obtenidas depende principalmente de los parámetros de la disolución. En medio acuoso, el glicósido sulfatado se liberó de las fibras más lentamente que el que no tenía dicho grupo funcional. La viabilidad de las células neurales no se vio afectada por los glicósidos. Conclusión: La preparación de microfibras alineadas de poliéster funcionalizadas con glicósidos es posible. La mayor parte del glicósido permanece retenido en las fibras sumergidas en agua después de varios días. El electrohilado es una técnica muy accesible y versátil para la fabricación de soportes en estrategias de terapia celular de lesiones medulares (AU)

Objective: Preparation of functionalized micro- and submicrofibers by electrospinning of polyesters with glycosides which are structural elements of proteoglycans, for application to the repair of spinal cord lesions. Material and methods: Solutions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with synthetic glycosides were prepared varying systematically the processing conditions. Fiber morphology assessed by scanning electron microscopy. The stability of the interaction between the glycoside and the polymer fiber was evaluated in aqueous medium, and their toxicity in cultures of neural cells. Results: The fiber morphology was altered mainly by the solution parameters. In aqueous medium, the glycoside with a sulfate group was released from fibers at slower rate than the non-sulfated glycoside. The viability of neural cells was not affected by the glycosides. Conclusion: It is possible to fabricate aligned polyester micro fibers with glycosides. Most of the glycoside present in the fibers remains in the substrate after extraction in water for several days. Electrospinning is a very accessible and versatile technique for application to strategies of cellular therapy in spinal cord injuries (AU)

Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors.

An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.

Cloning and overexpression of rhamnose isomerase and fucose isomerase.

Rhamnose isomerase and fucose isomerase were overexpressed in E. coli, purified and characterized. The rhamnose isomerase gene was ligated to the restriction sites of PstI and Hind III of vector pTrcHis and the fucose isomerase gene was ligated to the EcoRI and PstI sites of vector pKK223-3 for overexpression of the enzymes in E. coli XL1-Blue MRF. Approximately 16,500 U of active fucose isomerase and 2400 of rhamnose isomerase can be obtained per liter of culture from these expression systems.

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: an application of reverse screening.

X-ray quality crystals of class I-deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 183.1 A, b = 61.4 A, c = 49.3 A and a = 179.2 A, b = 60.5, A, c = 49.1 A, respectively. Two molecules in the asymmetric unit are related by a noncrystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 A. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

Cloning, overexpression and isolation of the type II FDP aldolase from E. coli for specificity study and synthetic application.

A stable overexpression E. coli strain containing the plasmid pKEN 2 for the production of the Zn(2+)-dependent FDP aldolase from E. coli has been developed. Approximately 14,000 U of the enzyme (specific activity = 23.3 U/mg) can be obtained from 4-L of growth medium. The enzyme was isolated, purified to homogeneity and used for the studies of stability, substrate specificity and metal ion replacement and dissociation. Crystals of the enzyme have been obtained for structural analysis. This E. coli strain was deposited with the American Type Culture Collection (ATCC #77472).

The use of immobilized cells to stabilize orsellinate depside hydrolase of Pseudevernia furfuracea.

Mixed cells of Pseudevennia Lun-Lunacea thallus have been entrapped in polyacrylamide gel and used in a continuous column process to hydrolyze evernic acid. Equimolar amounts of both orsellinic and everninic acids are recovered. The activity of entrapped cells, which contain orsellinate depside hydrolase, is unaltered over a period of two months.