High-performance affinity chromatography method for identification of L-arginine interacting factors using magnetic nanobeads.

L-Arginine exhibits a wide range of biological activities through a complex and highly regulated set of pathways that remain incompletely understood at both the whole-body and the cellular levels. The aim of this study is to develop and validate effective purification system for L-arginine interacting factors (AIFs). We have recently developed novel magnetic nanobeads (FG beads) composed of magnetite particles/glycidyl methacrylate (GMA)-styrene copolymer/covered GMA. These nanobeads have shown higher performance compared with commercially available magnetic beads in terms of purification efficiency. In this study, we have newly developed L-arginine methyl ester (L-AME)-immobilized beads by conjugating L-AME to the surface of these nanobeads. Firstly, we showed that inducible nitric oxide synthase, which binds and uses L-arginine as a substrate, specifically bound to L-AME-immobilized beads. Secondly, we newly identified phosphofructokinase, RuvB-like 1 and RuvB-like 2 as AIFs from crude extracts of HeLa cells using this affinity chromatographic system. The data presented here demonstrate that L-AME-immobilized beads are effective tool for purification of AIFs directly from crude cell extracts. We expect that the present method can be used to purify AIFs from various types of cells.

Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage.

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.

SV40 vectors carrying minimal sequence of viral origin with exchangeable capsids.

Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.

Induction of estrus by prostaglandin F2alpha administration in the vaginal vestibules of miniature pigs.

Estrus induction is an important step in embryo production. It has been difficult to induce estrus in miniature pigs by intramascular (i.m.) injection of prostaglandin F2alpha (PGF2(2alpha)) in the early luteal stage of the estrous cycle. In the present study, we injected two different doses of PGF2(2alpha) i.m. and into the submucosa of the vaginal vestibule (i.ves.) of miniature pigs, and examined the effect of these treatments on estrus induction. Fifteen miniature pigs were divided into five experimental groups (control, saline injected i.m.; PGF2alpha treated, 1.0 or 1.5 mg of PGF2alpha injected twice i.m. or i.ves.), and the estrus length and concentrations of 17beta-estradiol (E2) and progesterone (P) in the blood were examined. Estrus length was significantly shortened by a large amount of PGF2alpha injected i.ves. In addition, the concentration of P in the blood significantly decreased after two injections of PGF2alpha (i.m. or i.ves.). These results suggest that in miniature pigs, administration of at least 3.0 mg of PGF2alpha is required for the induction of luteolysis and injection of PGF2alpha into the vaginal vestibule is a useful method of estrus induction.