DiGeorge syndrome chromosomal region 8 (Dgcr8), a candidate gene for 22q11.2 deletion-associated schizophrenia, encodes an essential component for microRNA (miRNA) biosynthesis that plays a pivotal role in hippocampal learning and memory. Adult neurogenesis is known to be important in hippocampus-dependent memory, but the role and molecular mechanisms of adult neurogenesis in schizophrenia remain unclear. Here, we show that Dgcr8 heterozygosity in mice leads to reduced cell proliferation and neurogenesis in adult hippocampus, as well as impaired hippocampus-dependent learning. Several schizophrenia-associated genes were downregulated in the hippocampus of Dgcr8 ϩ/Ϫ mice, and one of them, insulin-like growth factor 2 (Igf2), rescued the proliferation of adult neural stem cells both in vitro and in vivo. Furthermore, IGF2 improved the spatial working memory deficits in Dgcr8 ϩ/Ϫ mice. These data suggest that defective adult neurogenesis contributes to the cognitive impairment observed in 22q11.2 deletion-associated schizophrenia and could be rectified by IGF2.

The JEOL Automated Data Acquisition System (JADAS) is a software system built for the latest generation of the JEOL Transmission Electron Microscopes. It is designed to partially or fully automate image acquisition for ice-embedded single particles under low dose conditions. Its builtin flexibility permits users to customize the order of various imaging operations. In this paper, we describe how JADAS is used to accurately locate and image suitable specimen areas on a grid of iceembedded particles. We also demonstrate the utility of JADAS by imaging the epsilon 15 bacteriophage with the JEM3200FSC electron cryo-microscope, showing that sufficient images can be collected in a single 8-hour session to yield a subnanometer resolution structure which agrees with the previously determined structure.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

No abstract

Triterpenoids are a group of structurally diverse specialized metabolites that frequently show useful bioactivities. These chemicals are biosynthesized from the common precursor 2,3-oxidosqualene in plants. The carbon skeletons produced by oxidosqualene cyclase (OSC) are usually modified by cytochrome P450 monooxygenases (P450s) and UDP-dependent glycosyltransferases. These biosynthetic enzymes contribute to the structural diversification of plant triterpenoids. Until now, many P450 enzymes have been characterized as triterpenoid oxidases. Among them, the CYP716 family P450 enzymes, which have been isolated from a wide range of plant families, seem to contribute to the triterpenoid structural diversification. Many CYP716 family P450 enzymes have been characterized as the multifunctional triterpene C-28 oxidases, which oxidize α-amyrin and β-amyrin to the widely distributed triterpenoids ursolic and oleanolic acids, respectively. Tomato (Solanum lycopersicum) is one of the most important solanaceous crops in the world. However, little information is known regarding its triterpenoid biosynthesis. To understand the mechanism of triterpenoid biosynthesis in tomato, we focused on the function of CYP716 family enzymes as triterpenoid oxidases. We isolated all six CYP716 family genes from the Micro-Tom cultivar of tomato, and functionally characterized them in the heterologous yeast expression system. The in vivo enzymatic assays showed that CYP716A44 and CYP716A46 exhibited the ordinary C-28 oxidation activity against α-amyrin and β-amyrin to produce ursolic and oleanolic acids, respectively. Interestingly, one CYP716E subfamily enzyme, CYP716E26, exhibited the previously unreported C-6β hydroxylation activity against β-amyrin to produce a rare bioactive triterpenoid, daturadiol (olean-12-ene-3β,6β-diol). To determine the roles of the CYP716 family genes in tomato triterpenoid biosynthesis, we analyzed the gene expression and triterpenoid accumulation patterns in different plant tissues by performing the quantitative real-time polymerase chain reaction (qPCR) and gas chromatography-mass spectrometry (GC-MS) analyses, respectively. High levels of the CYP716A44 gene expression and the accumulation of C-28-oxidized triterpenoids, ursolic acid, and oleanolic acid were observed in the roots, indicating a significant contribution of the CYP716A44 gene in the triterpenoid biosynthesis in tomato. Thus, our study partially elucidated the mechanism of triterpenoid biosynthesis in tomato, and identified CYP716E26 as a novel C-6β hydroxylase for its subsequent use in the combinatorial biosynthesis of bioactive triterpenoids.