Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Characterization of protein hydrolysates of cosmetic use by CE-MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid - pH 1.8 - in 70: 30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75: 25-25: 75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.

Synthesis, Spectroscopic Studies and X-ray Crystal Structures of New Pyrazoline and Pyrazole Derivatives

The synthesis and characterization of some pyrazoline compounds of 1,3-diketones with hydrazine derivatives, namely, 1-(S-benzyldithiocarbazate)-3-methyl-5-phenyl-5-hydroxypyrazoline (1); 1-(2-thiophenecarboxylic)-3-methyl-5-phenyl-5-hydroxypyrazoline (2); 1-(2-thiophenecarboxylic)-3,5-dimethyl-5-hydroxypyrazoline (3); 1-(S-benzyldithiocarbazato)-3-methyl-5-phenylpyrazole (4); 1-(2-thiophenecarboxylic)-3-methyl-5-phenylpyrazole (5) and 1-(S-benzyldithiocarbazate)-3,5-dimethylpyrazole (6) are reported. Studies by IR, ((1)H, (13)C)-NMR spectroscopies and single crystal X-ray diffraction revealed that compounds (1)(,) (2) and (3) are formed as pyrazoline, whereas (4) and (5) are formed as pyrazole derivatives only under acidic conditions. Compound (1) crystallizes in orthorhombic P2(1)2(1)2(1), a = 6.38960(10) angstrom, b = 12.9176(3) angstrom, c = 21.2552(5) angstrom, (2) crystallizes in monoclinic, P2(1)/n, a = 11.3617(2) angstrom, b = 8.4988(2) angstrom, c = 92.8900(10)angstrom and beta = 92.8900(5)degrees, (3) crystallizes in monoclinic, C2/c, a = 15.9500(5) angstrom, b = 9.3766(3) angstrom, c = 16.6910(5)angstrom and beta = 113.825(2)degrees, (4) crystallizes in monoclinic, P2(1)/c, a = 15.228(4) angstrom, b = 5.5714(13) angstrom, c = 19.956(5)angstrom and beta = 91.575(7)degrees and (6) crystallizes in orthorhombic, P2(1)2(1)2(1), a = 5.3920(2) angstrom, b = 11.2074(5) angstrom, c = 21.885(1)angstrom . The (3) derivative represents the first pyrazoline compound prepared from 2,4-pentanedione and characterized crystallographically.