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Increasing evidence indicates that many insecticides produce significant epigenetic changes during embryogenesis, leading to developmental toxicities. However, the effects of insecticides on DNA methylation status during early development have not been well studied. We developed a novel nuclear phenotypic approach using mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation changes via high-content imaging analysis. Exposure to imidacloprid, carbaryl, and o,p'-DDT increased the fluorescent intensity of granules in the nuclei, indicating global DNA methylating effects. However, DNA methylation profiling in cell-cycle-related genes, such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, decreased in imidacloprid treatments, suggesting the potential influence of DNA methylation patterns on cell differentiation. We developed a rapid method for evaluating global DNA methylation and used this approach to show that insecticides pose risks of developmental toxicity through DNA methylation.

Piceatannol (3,3',4',5-trans-tetrahydroxystilbene) is a polyphenolic compound abundant in the seeds of passion fruit (Passiflora edulis). Piceatannol is an analogue of resveratrol (3,4',5-trans-trihydroxystilbene) and shares the structural motif and biological activities such as activation of SIRT1. Several studies have shown that piceatannol is more potent than resveratrol. In this study, we examined the effects of piceatannol on neural stem cell differentiation into astrocytes compared with those of resveratrol. At a concentration of 2.5 μM, piceatannol promoted astrocyte differentiation, while resveratrol had no effect at this concentration. Furthermore, we found that oral administration of piceatannol increased the number of astrocytes in the brains of adult mice, while resveratrol administration showed no effects. These results suggest that piceatannol has a superior effect to resveratrol in promoting astrocyte differentiation.

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three β-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey's method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Kruppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in expressing various neural functions including memory consolidation. Alterations of BDNF levels in the brain are associated with neurodegenerative and neuropsychiatric disorders. Therefore, it is important to understand how levels of BDNF are controlled. Recently we generated a novel transgenic mouse strain, termed the Bdnf-Luciferase transgenic (Bdnf-Luc Tg) mouse, to monitor changes in Bdnf expression. In the present study, we detected the bioluminescence signal from living Bdnf-Luc Tg mice after intraperitoneal administration of d-luciferin. Despite high levels of Bdnf expression in the brain, it was difficult to detect a signal from the brain region, probably because of its poorly penetrable (short-wavelength) bioluminescence. However, we could detect the changes in the bioluminescence signal in the brain region using a luciferin analogue generating a near-infrared wavelength of bioluminescence. We also found a strong correlation between increases in body weight and bioluminescence signal in the abdominal region of Tg mice fed a high-fat diet. These results show that changes in Bdnf expression can be visualized using living mice, and that the Tg mouse could be a powerful tool for clarification of the role of Bdnf expression in pathophysiological and physiological conditions.

A bio-inspired cascade reaction has been developed for the construction of the marine natural product ageladine A and a de novo array of its N1-substituted derivatives. This cascade features a 2-aminoimidazole formation that is modeled after an arginine post-translational modification and an aza-electrocyclization. It can be effectively carried out in a one-pot procedure from simple anilines or guanidines, leading to structural analogues of ageladine A that had been otherwise synthetically inaccessible. We found that some compounds out of this structurally novel library show a significant activity in modulating the neural differentiation. Namely, these compounds selectively activate or inhibit the differentiation of neural stem cells to neurons, while being negligible in the differentiation to astrocytes. This study represents a successful case in which the native biofunction of a natural product could be altered by structural modifications.

A mouse cDNA encoding paired-box-containing gene (Pax-4) was cloned from a cDNA library of a mouse pancreatic islet β cell line of MIN6. The predicted open reading frame encodes a protein of 349 amino acids with a calculated molecular weight (MW) of 38-kD. A human Pax-4 cDNA sequence encoding 350 amino acids was deduced from a human cosmid clone. The mouse nucleotide and deduced amino acid sequences exhibit 83.4 and 80.0% identity with those of deduced human Pax-4, respectively. Southern blot analysis suggested that the mouse Pax-4 gene exists as a single copy in the genome. Reverse transcription (RT)-PCR analysis suggested that the mouse Pax-4 gene is expressed in pancreatic islets, cultured islet β cell lines of MIN6, βTC, and NIT-1 cells, but not detectable in any of 13 adult mouse organs examined.

A cDNA clone encoding a human ribosomal protein L39 (hRPL39) was isolated through a random cDNA sequencing approach to a cDNA library constructed from a human colon carcinoma cell line of COLO 205. Although levels of hRPL39 mRNA were different in several cell lines including carcinoma cell lines from different tissues, they were shown not to be cell cycle-dependent in a human fibroblast cell line of TIG-1.