Absorción mineral y retención ósea en ratas normales en crecimiento por el consumo de un yogur experimental reducido en lactosa que contiene galactooligosacáridos (GOS) / Mineral absorption and bone retention in normal growing rats by consumption of an experimental lactose-reduced yogurt containing galactooligosaccharides (GOS)

Introducción: Los GOS son prebióticos naturales presentes en la leche materna que pue-den obtenerse enzimáticamente a partir de la lactosa de leche de vaca durante la fabricación de yogur. El producto lácteo resultante será reducido en lactosa y contendrá prebióticos y bacterias potencialmente probióticas. Sin embargo, mantendrá la baja relación Ca/Pi que aporta la leche de vaca, lo que podría alterar el remodelamiento óseo y la mineralización. Objetivo: comparar si un yogur reducido en lactosa que contiene GOS (YE) ofrece ventajas adicionales respecto de un yogur regular sin GOS (YR) sobre las absorciones (Abs) de Ca y Pi, retención y calidad ósea durante el crecimiento normal. Al destete, ratas machos fueron divididas en 3 grupos alimentados con AIN ́93-G (C), YE o YR durante 28 días. Resultados: YE mostró el mayor aumento de lactobacilos fecales; producción de ácidos grasos de cadena corta especialmente p, profundidad de las criptas colónicas y menor pH cecal. El %AbsCa y %AbsPi aumentó en el siguiente órden: YE> YR> C (p < 0,05). El contenido de Ca y Pi en fémur, la densidad y contenido mineral óseos y los parámetros biomecánicos fueron similares en YE y C, mientras que YR mostró valores significativa-mente menores (p < 0,05). Conclusiones: YE aumentó las Abs y biodisponibilidad de minerales, alcanzando la retención y calidad ósea de C. El aumento en las Abs observado en YR no logró obtener la retención y calidad ósea de C. Conclusión: YE habría contrarrestado el efecto negativo del mayor aporte de Pi de la leche de vaca y sería una buena estrategia para lograr el pico de masa ósea y calidad del hueso adecuados, especialmente en individuos intolerantes a la lactosa. (AU)

Breast milk contains an optimal calcium/phosphate (Ca/Pi) ratio and GOS. These natural prebiotics can be enzymatically produced via cow's milk lactose inyogurt manufacture. This milk product is low in lactose and contains prebiotics and potentially probiotic bacteria but maintains a low Ca/Pi ratio that could alter bone remodeling and mineralization. We evaluated if a lactose-reduced yogurt containing GOS (YE) offers additional advantages over regular yogurt without GOS (YR) on Ca and Pi absorption (Abs), bone retention and quality during normal growth. Weaning male rats were divided into 3 groups fed AIN'93-G (C), YE or YR for 28 days. Results: YE showed the highest increase in fecal lactobacilli; short-chain fatty acids production, especially propionate and butyrate; intestine crypt depth, and the lowest cecal pH. AbsCa% and AbsPi% increased in this order: YE> YR> C (p <0.05). Ca and Pi content in femur, bone density and mineral content, and biomechanical parameters were similar in YE and C, while YR showed the significantly lowest value (p < 0.05). Conclusions: YE increased mineral Abs reaching the retention and bone quality of C. Although YR increased Abs, bone retention and quality did not achieve C values. Seemingly, YE compensated for the negative effect of the higher Pi supply and would be a good strategy to achieve adequate peak bone mass and bone quality, especially in lactose intolerant individuals. (AU)

Enzymatic Synthesis of 2-(ß-Galactosyl)-ethyl Methacrylate by ß-Galactosidase from Pyrococcus woesei and Application for Glycopolymer Synthesis and Lectin Studies.

Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(ß-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed KD values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates.

Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.

Anti-tumor activity of cytotoxic T lymphocytes elicited with recombinant and synthetic forms of a model tumor-associated antigen.

The recent cloning of tumor-associated antigens (TAAs) recognized by CD8+ T lymphocytes (TCD8+) has made it possible to use recombinant and synthetic forms of TAAs to generate TCD8+ with anti-tumor activity. To explore new therapeutic strategies in a mouse model, we retrovirally transduced the experimental murine tumor CT26(H-2d), with the lacZ gene encoding our model TAA, beta-galactosidase (beta-gal). The transduced cell line, CT26.CL25, grew as rapidly and as lethally as the parental cell line in normal, immunocompetent animals. In an attempt to elicit TCD8+ directed against our model TAA by using purely recombinant and synthetic forms of our model TAA, we synthesized a nine-amino-acid long immunodominant peptide of beta-gal (TPH-PARIGL), corresponding to amino acid residues 876-884, which was known to be presented by the Ld major histocompatibility complex (MHC) class I molecule, and a recombinant vaccinia virus encoding the full-length beta-gal protein (VJS6). Splenocytes obtained from naïve mice and co-cultured with beta-gal peptide could not be expanded in primary ex vivo cultures. However, mice immunized with VJS6, but not with a control recombinant vaccinia virus, yielded splenocytes that were capable of specifically lysing CT26.CL25 in vitro after co-culture with beta-gal peptide. Most significantly, adoptive transfer of these cells could effectively treat mice bearing 3-day-old established pulmonary metastases. These observations show that therapeutic TCD8+ directed against a model TAA could be generated by using purely recombinant and synthetic forms of this antigen. These findings point the way to a potentially useful immunotherapeutic strategy, which has been made possible by the recent cloning of immunogenic TAAs that are expressed by human malignancies.

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.