4H-1,3-Oxazin-4-one誘導体の環変換反応を利用したAzaphenanthrene誘導体の合成

Reactions of 4H-1, 3-oxazin-4-ones with cyclic ketones such as α-tetralone, β-tetralone, and Δ^-octalone-2 afforded 9, 10-dihydrobenz [f] isoquinolin-3(2H)-ones, 5, 6-dihydrobenz- [h] isoquinolin-3(2H)-ones, and 6, 7, 8, 9, 10-pentahydrobenz [g] isoquinolin-3(2H)-ones, respectively

Upregulation of Mir-21 Levels in the Vitreous Humor Is Associated with Development of Proliferative Vitreoretinal Disease.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by post-transcriptional inhibition of mRNA translation. Dysregulation of miRNAs, including circulating miRNAs, has been reported to play an important role in the development of various diseases, including fibrotic diseases. Aberrant expression of miRNAs in the vitreous humor of vitreoretinal diseased eyes has been reported. However, the expression pattern of miRNAs present in the vitreous humor of proliferative vitreoretinal disease (PVD) patients, including proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), remains unknown. To investigate the factors important for the development of PVD, we characterized the miRNAs present in the vitreous humor of PVD patients and analyzed the expression profiles of 377 miRNAs using quantitative polymerase chain reaction-based miRNA arrays. The expression of a specific subset of miRNAs, previously reported to be associated with the development of angiogenesis and fibrosis, was significantly altered in the vitreous of PVD patients. Among these miRNAs, we identified miR-21 as a candidate fibrotic miRNA with an important role in the pathogenesis of PVD. Increased miR-21 levels in the vitreous were associated with retinal fibrosis, including PVR and PDR. Because epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPECs) plays a critical role in retinal fibrosis, the expression of miR-21 in human RPECs was determined. Its expression in RPECs was induced by transforming growth factor-β, a key growth factor involved in fibrogenesis, and was enhanced by high glucose culture conditions, suggesting that miR-21 expression positively correlates with disease progression. Gain- and loss-of-function studies revealed that miR-21 promoted cell proliferation and migration of ARPE-19 cells without affecting EMT-related gene expression. Together, our studies have identified miR-21 as a potential disease-modifying miRNA in the vitreous humor that is involved in the development of retinal fibrosis and may be a novel marker of PVD

Expression patterns of zebrafish heterochronic genes during embryonic development.

<p>A. Semiquantitative RT-PCR analysis of <i>lin-28a, lin-28b and lin-41</i> gene mRNA expression. Total RNA was isolated from wild-type embryos at the indicated developmental stages and analyzed by using specific primers for <i>lin-28a</i>, <i>lin-28b</i> and <i>lin-41</i>. <i>gapdh</i> was used as a control. B. Real-time PCR analysis of <i>let-7a</i>, <i>let-7b</i> and <i>lin-4/miR-125b</i> miRNA expression. Total RNA was isolated from wild-type embryos at the indicated developmental stages. Expression levels of <i>let-7a</i>, <i>let-7b</i> and <i>lin-4/miR-125b</i> was analyzed using TaqMan miRNA assay. C. Developmental expression analysis of zebrafish <i>lin-28a</i> and <i>lin-28b</i> mRNAs by whole mount in situ hybridization. Zebrafish embryos at various stages were hybridized with antisense probes. D, E. Developmental expression analysis of zebrafish <i>lin-41</i> mRNAs by whole mount in situ hybridization. <i>sox2</i> and <i>otx2</i> probes were used as a CNS maker. Arrowhead indicated expression in the CNS.</p

Expression of miRNAs and downstream heterochronic genes in <i>lin-28a</i> and <i>lin-28b</i> morphant embryos.

<p>A. miRNA expression profiling in <i>lin-28a</i> and <i>lin-28b</i> morphants embryos at 5 hpf. Hierarchically clustered heat-map representing differences in miRNA expression between control MO-injected embryos and <i>lin-28a</i> MO- or <i>lin-28b</i> MO-injected embryos. Relative levels of expression are colored from green (low) to red (high). B. Real-time PCR analysis of <i>let-7a, let-7b, miR-430a</i> and <i>miR-430b</i> expression in control MO-, <i>lin-28a</i> MO- and <i>lin-28b</i> MO-injected embryos. Expression levels of <i>let-7a, let-7b, miR-430a</i> and <i>miR-430b</i> were analyzed at 5 hpf using TaqMan miRNA assay. The expression level of <i>miR-430a</i> and <i>miR-430b</i> was downregulated in both <i>lin-28a</i> MO- and <i>lin-28b</i> MO-injected embryos compared with control MO-injected embryos (***p<0.001, control MO vs. <i>lin-28a</i> MO, control MO vs. <i>lin-28b</i> MO, two-tailed test, n = 3, mean ± SEM). C. Real-time PCR analysis of <i>let-7a</i> and <i>let-7b</i> expression in control MO-, lin-28a MO- and lin-28b MO-injected embryos. Expression levels of <i>let-7a</i> and <i>let-7b</i> were analyzed using TaqMan miRNA assay at 28 hpf. The expression level of <i>let-7a</i> and <i>let-7b</i> was increased in both <i>lin-28a</i> MO- and <i>lin-28b</i> MO-injected embryos embryos compared with control MO-injected embryos (*p<0.05, **p<0.01, control MO vs. <i>lin-28a</i> MO, control MO vs. <i>lin-28b</i> MO, two-tailed test, n = 4, mean ± SEM). D. Semiquantitative RT-PCR analysis of <i>lin-41</i> gene expression in control MO-, lin-28a MO- and lin-28b MO-injected embryos. Total RNA was isolated from <i>lin-28a</i> MO-, <i>lin-28b</i> MO- or control MO-injected embryos at 24 hpf and analyzed by using specific primers for <i>lin-41.</i> The expression of <i>lin-41</i> was decreased in both <i>lin-28a</i> MO- and <i>lin-28b</i> MO-injected embryos compared with control MO-injected embryos. E. Control, <i>lin-28a</i> and <i>lin-28b</i> MO-injected embryos labeled by <i>in situ</i> hybridization with <i>lin-41</i> probe. Expression of <i>lin-41</i> was repressed in CNS of both <i>lin-28a</i> and <i>lin-28b</i> morphant embryos at 24 hpf.</p

Cloning and characterization of zebrafish Lin-28a and Lin-28b.

<p>A. Deduced amino acid sequences of zebrafish Lin-28a and Lin-28b aligned against human, mouse and c.elegans Lin28 homologs. Blue boxes represent identical and similar amino acids. The characteristic Lin28 structure including cold-shock domain and two CCHC-Zn finger domains was highly conserved in zebrafish Lin-28a and Lin-28b. B. The phylogeny of the Lin28 family based on the alignment of full length of amino acid sequence. The branch length (X axis) in the rectangular cladogram represent the distances among those sequences calculated using BLOSUM62 substitution matrix.</p

Morpholino-mediated knockdown of <i>lin-28a</i> and <i>lin-28b</i> affects early development.

<p>A. B. Schematic representation shows the genomic organization of the <i>lin-28a</i> and <i>lin-28b</i> genes in zebrafish. Regions targeted by splice-blocking morpholinos are shown. Arrows depict the location of the two primer sets to amplify exon and intron sequences, which were utilized in the RT-PCR analysis. C. D. The efficacy of MO was validated by RT-PCR using primers as indicated in A, B. Total RNA was isolated from <i>lin-28a</i>, <i>lin-28b</i> or control MO-injected embryos at 5 hpf and analyzed by using specific primers for <i>lin-28a</i> and <i>lin-28b</i>. Upper panel shows a reduced expression of <i>lin-28a</i> and <i>lin-28b mRNA</i>, and an increase of an aberrantly spliced <i>lin-28b</i> transcripts corresponding to the presence of intron 1. Lower panel shows RT-PCR products with <i>gapdh</i> primers used as internal control. E. Representative images of severe phenotype of <i>lin-28a</i> and <i>lin-28b</i> morphants at 5 hpf. F. Vegetal view of control, <i>lin-28a</i> and <i>lin-28b</i> morphants at 8 hpf. Arrow shows the similar extent of prechordal plate extension in control, <i>lin-28a</i> and <i>lin-28b</i> morphant embryos; bracket shows a reduced extent in epiboly in <i>lin-28a</i> and <i>lin-28b</i> morphant embryos compared with controls. G. Representatives of mild and severe phenotypes in <i>lin-28a</i> and <i>lin-28b</i> morphants at 30 hpf. Both <i>lin-28a</i> and <i>lin-28b</i> morphants develop morphological phenotypes displaying shorten body axis, small anterior structures and aberrant tail morphology ranging from mild (Right) to severe (Left) compare to control MO injected embryos (Top). Arrowheads indicate the smaller head. H. Quantification of the efficiency of rescue from gastrulation defects following co-injection of <i>lin-28a</i> MO, <i>lin-28b</i> MO and mRNAs. More than half of the <i>lin-28a</i> MO or <i>lin-28b</i> MO injected embryos had died by 30 hpf. The frequencies of the phenotypes were similar between <i>lin-28a</i> and <i>lin-28b</i> morphants. The frequency of the dead, mild and severe phenotypes decreased with a rescue experiment in which 300 pg of <i>lin-28a</i> and <i>lin-28b</i> mRNAs were co-injected with the MO. (p<0.0001, <i>lin-28a</i> MO vs. <i>lin-28a</i> MO+<i>lin-28a</i> mRNA, <i>lin-28b</i> MO vs. <i>lin-28b</i> MO+<i>lin-28b</i> mRNA, Fisher’s exact test). The total number of embryos is noted above each bar.</p