Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

Structural Features of Transcription Factors Associating with Nucleosome Binding.

Fate-changing transcription factors (TFs) scan chromatin to initiate new genetic programs during cell differentiation and reprogramming. Yet the protein structure domains that allow TFs to target nucleosomal DNA remain unexplored. We screened diverse TFs for binding to nucleosomes containing motif-enriched sequences targeted by pioneer factors in vivo. FOXA1, OCT4, ASCL1/E12α, PU1, CEBPα, and ZELDA display a range of nucleosome binding affinities that correlate with their cell reprogramming potential. We further screened 593 full-length human TFs on protein microarrays against different nucleosome sequences, followed by confirmation in solution, to distinguish among factors that bound nucleosomes, such as the neuronal AP-2α/ß/γ, versus factors that only bound free DNA. Structural comparisons of DNA binding domains revealed that efficient nucleosome binders use short anchoring α helices to bind DNA, whereas weak nucleosome binders use unstructured regions and/or ß sheets. Thus, specific modes of DNA interaction allow nucleosome scanning that confers pioneer activity to transcription factors.

Baeyer-Villiger reaction of steroid sapogenins by CF3COOH-H2O2. A short cut to pregnan-3ß,16ß,20-triol 3-monoacetates.

Treatment of steroid sapogenins with H2O2 in CF3COOH for 15min followed by reflux in CH3OH/H2O afforded good yields of pregnan-3ß,16ß,20-triol 3-monoacetates. When the hydrolysis step was carried out with KOH in refluxing methanol excellent yields pregnantriols were obtained. The resulting compounds were characterized by their melting points and NMR spectral data. An X-ray diffraction analysis of compound 3a confirmed the proposed structure and provided detailed information about the bond lengths, bond angles and conformation.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.(AU)

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.(AU)

Caracterización electroforética y cromatográfica del veneno del alacrán Rhopalurus junceus / Electrophoretic and chromatographic characterization of the Rhopalurus junceus scorpion venom

El veneno del alacrán azul, Rhopalurus junceus es actualmente comercializado con el nombre de Escozul. Este producto natural se emplea en el tratamiento de diferentes patologías, sin embargo en la literatura revisada no aparece información referente a la caracterización bioquímica del extracto de este ejemplar cubano. Objetivo: hacer una caracterización bioquímica preliminar del veneno crudo del alacrán cubano mediante el empleo de dos técnicas destinadas a estos fines. Método: se empleó la electroforesis discontinua de proteínas en gel de poliacrilamida al 15% y la cromatografía de alta presión. Resultados: se identificó un patrón difuso correspondiente a péptidos de talla inferiores a catorce KDa, mientras que por HPLC se determinaron trece picos en el veneno crudo de esta especie. Conclusiones: el veneno del alacrán azul presenta una composición molecular similar a la descrita para otras especies con una mezcla molecular inferior a catorce KDa.

The blue scorpion venom, Rhopalurus junceus is currently marketed with the name of Escozul. This natural product is used in the treatment of different pathologies; however in the revised literature doesn't appear concerning information to the biochemical characterization of the extract of this Cuban specimen. Objective: to make a preliminary biochemical characterization on the crude venom of the Cuban scorpion by means of the employment of two techniques dedicated to these purposes. Method: the discontinuous electrophoresis of proteins was used in polyacrylamide to 15% and the chromatography of high pressure. Results: a diffuse pattern corresponding to inferior size peptides to fourteen KDa was identified, while for HPLC thirteen peaks were determined in the crude venom of this species. Conclusions: the blue scorpion venom presents a similar molecular composition to the one described for other species with an inferior molecular mixture to fourteen KDa.

Caracterización electroforética y cromatográfica del veneno del alacrán Rhopalurus junceus / Electrophoretic and chromatographic characterization of the Rhopalurus junceus scorpion venom

El veneno del alacrán azul, Rhopalurus junceus es actualmente comercializado con el nombre de Escozul. Este producto natural se emplea en el tratamiento de diferentes patologías, sin embargo en la literatura revisada no aparece información referente a la caracterización bioquímica del extracto de este ejemplar cubano. Objetivo: hacer una caracterización bioquímica preliminar del veneno crudo del alacrán cubano mediante el empleo de dos técnicas destinadas a estos fines. Método: se empleó la electroforesis discontinua de proteínas en gel de poliacrilamida al 15% y la cromatografía de alta presión. Resultados: se identificó un patrón difuso correspondiente a péptidos de talla inferiores a catorce KDa, mientras que por HPLC se determinaron trece picos en el veneno crudo de esta especie. Conclusiones: el veneno del alacrán azul presenta una composición molecular similar a la descrita para otras especies con una mezcla molecular inferior a catorce KDa (AU)

The blue scorpion venom, Rhopalurus junceus is currently marketed with the name of Escozul. This natural product is used in the treatment of different pathologies; however in the revised literature doesn't appear concerning information to the biochemical characterization of the extract of this Cuban specimen. Objective: to make a preliminary biochemical characterization on the crude venom of the Cuban scorpion by means of the employment of two techniques dedicated to these purposes. Method: the discontinuous electrophoresis of proteins was used in polyacrylamide to 15% and the chromatography of high pressure. Results: a diffuse pattern corresponding to inferior size peptides to fourteen KDa was identified, while for HPLC thirteen peaks were determined in the crude venom of this species. Conclusions: the blue scorpion venom presents a similar molecular composition to the one described for other species with an inferior molecular mixture to fourteen KDa (AU)