Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 degrees C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9 h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 degrees C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.     

Amyloid-beta fibril formation is not necessarily required for microglial activation by the peptides

There is increasing evidence that microglial activation has pathogenic influence on Alzheimer's disease. According to in vitro studies, microglia activated by amyloid-beta (Abeta) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, nitric oxide or reactive oxygen species. Although the relationship between the aggregational state of Abeta peptides and their neurotoxic activities has been well investigated, little is known about the relationship between the aggregational state of Abeta peptides and their ability to induce microglial activation. In the present study, we thus performed both structural and biochemical studies to clarify the relationship between the aggregational state of Abeta peptides and their ability to activate microglia. Our results have shown that, in the presence of interferon-gamma, the Abeta25-35(M(35)Nle) peptide had almost the same potency of activating microglia and producing TNF-alpha as the Abeta25-35 peptide on both protein and mRNA levels, in spite of the fact that former peptide represented much less amyloid fibril formation than the latter in a thioflavine-T fluorometric assay. These results suggest that Abeta fibril formation is not necessarily required for microglial activation by the peptides.     

Maleimide derivative of hapten for coupling to enzyme: a new method in enzyme immunoassay.

Meta-maleimidobenzoyl derivative of L-thyroxine methyl ester (MBTM) was synthesized and coupled to β-galactosidase at molar ratio of over 5 to 1. More than 97% of the enzyme was found to be labeled with MBTM as examined by double antibody precipitation method in excess of anti-T4 antibody. Maleimide group of MBTM was found to be labile; about 50% was destroyed in 3 hours when prepared in a solution of 1 μg/ml phosphate buffer (pH 7.0, 0.05M). With antiserum dilution of 2,400 fold, reproducible T4 enzyme immunoassay was carried out using double antibody precipitation method. A high sensitivity in the assay was observed on the 0–10 μg/100 ml range.