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.

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.

Copy numbers of alpha amylase genes (AMY), which encode starch-digesting enzymes, are markedly increased in modern humans and domesticated dogs as an adaptive evolutionary mechanism in response to increased consumption of starch-rich foods acquired either by farming or domestication. In this study, we surveyed total AMY gene copy numbers in 150 domestic pigs (50 pigs of Berkshire breed, 50 of Landrace breed, and 50 of Large White breed) and 51 wild boars (30 Sus scrofa leucomystax and 21 S. s. riukiuanus) to identify whether the gene copy number has changed during the domestication of pigs. The relative copy number of AMY genes was measured using a quantitative polymerase chain reaction (qPCR) and it varied from 2.7 to 10.8 per haploid genome among individuals. However, in the four remaining populations, excluding S. s. riukiuanus, the average copy number was approximately six, and no significant differences were observed between the three selected pig breeds and S. s. leucomystax wild boar. Conversely, S. s. riukiuanus had an average of 7.2 copies. The results indicating six AMY copies per haploid genome were consistent with the porcine genome reference sequence (Sscrofa11.1). These results suggest that there has been no significant increase in the AMY gene copy number during the domestication process of pigs.