We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2 ؉/؊ ; Crx ؊/؊ retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx ؊/؊ retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2 ؉/؊ ; Crx ؊/؊ retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cellspecific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.The vertebrate neural retina is comprised of six types of neurons and one type of glial cell, all derived from one population of multipotent progenitors (38,39,41). Transcription factors such as homeobox and basic helix-loop-helix factors have been known to play pivotal roles in the specification and development of retinal cell subtypes. Among the Otx-like homeobox genes, Otx2 and Crx play critical roles in retinal photoreceptor development. The expression of Otx2 covers most of the forebrain and midbrain neuroepithelium, including the eye domain, during development (32). Complete elimination of Otx2 functions in mice by gene targeting results in the absence of the forebrain and embryonic lethality (1,3,27). In a previous study, we have shown that Otx2 is essential and sufficient for the cell fate determination of retinal photoreceptors (29). Crx, on the other hand, is reported to be expressed abundantly in retinal photoreceptors and pinealocytes and also weakly in retinal bipolar cells (12,19). It has also been reported that Crx regulates various photoreceptor-specific genes (12,19,20). Mutations of human CRX are associated with three types of photoreceptor diseases: cone-rod dystrophy 2, retinitis pigmentosa, and Leber's congenital amaurosis (15,16,33,36). A gene-targeting study has revealed that Crx is essential for the terminal differentiation of photoreceptors and normal circadian entrainment (20). Thus, Otx2 and Crx have distinct roles in retinal photoreceptor development although their expression patterns in the retina overlap to some extent. However, they are structurally related transcription factors and can bind to a common DNA-bindin...

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) ⑀ as an effector of Ras. Here we have examined the role of PLC⑀ in de novo skin chemical carcinogenesis by using mice whose PLC⑀ is genetically inactivated. PLC⑀ ؊/؊ mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLC⑀ ؊/؊ mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLC⑀ ؉/؉ mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLC⑀ background. The skin of PLC⑀ ؊/؊ mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLC⑀ in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLC⑀ as a candidate molecular target for the development of anticancer drugs.

Leinamycin (1) was isolated from the culture broth of a Streptomyces sp. in 1989, 1-3 and its structure was elucidated by spectroscopic analysis, 1 X-ray crystallography, 4 and chemical synthesis. 5 This antibiotic contains an unusual 1,3-dioxo-1,2dithiolane moiety, which is connected to the 18-membered lactam through a spiro linkage, and appeared to be a new class of natural product. 1 exhibited significant antitumor activity in some murine tumor models. 2 We previously reported that 1 induces single-strand scission of DNA in the presence of thiol cofactors in vitro. We report here the detailed chemistry of thiol-activation and DNA-cleavage induced by 1.Addition of 1.5 equiv of 2-mercaptoethanol (2-ME) to a solution of 1 in MeOH/10 mM phosphate buffer (pH 7) (1/9, v/v) resulted in a rapid conversion to a major product with several minor products. Other thiols including ethanethiol, dithiothreitol, cysteine, and glutathione afforded approximately equal amounts of 2. We isolated 2 from the reaction mixture with reverse-phase HPLC (70%, yield), but the full characterization of 2 failed due to its instability in DMSO. Treatment of 2 with K 2 CO 3 and iodomethane afforded a stable methyl ester 2a (68%, yield). Characterization of 2a by 1D and 2D-NMR experiments established an unexpected structure, in which the 1,3-dioxo-1,2-dithiolane moiety and the 6,7-olefin were missing and a new 3,7-sulfide linkage and 6-hydroxyl group were observed. Treatment of 1 with 2-ME in a MeOH-rich solvent, MeOH/0.5 M phosphate buffer (pH 7) (99/1, v/v), afforded the methanol adduct 2b 6 as the main product.After reaction of 1 with calf thymus DNA in the presence of 2-ME (drug/DNAbp/2ME ) 1/20/1.5), the DNA was purified by ethanol precipitation. The DNA showed a UV spectrum characteristic of the complex with a chromophore. Although it was stable at 4°C, gradual release of the chromophore from the DNA was observed at 37°C. The rate and efficiency of the release increased with further an increase in the temperature. On a preparative scale, we isolated the released chromophore 3 from the leinamycin-treated calf thymus DNA (75% yield from 1). In the 13 C NMR spectrum of 3 all of the resonances were comparable to those of 2, and five additional resonances were found, suggesting the addition of a purine residue to 2. In the 1 H-NMR spectrum, all of the resonances were also comparable to those of 2. One additional nonexchangeable resonance at 7.77 ppm (1H) was found. The only nucleobase that gives one nonexchangeable resonance is guanine. Observation of the NOE between the guanine H-8 and the 6-CH 3 is consistent with alkylation of the C-6 carbon by the N-7 of guanine. These spectroscopic data revealed that 3 is a leinamycin-N7 guanine adduct. This was supported by the molecular formula that was established as the sodium salt C 27 H 31 O 7 N 7 S 2 Na: HRFABMS (M + Na) + m/z 652.1600, calcd 652.1624. 1 did not react with guanosine nucleotide monomer or single stranded DNA, suggesting that the alkylation of DNA could be attributable to the unique...

BACKGROUND AND PURPOSEAnticonvulsants have been developed according to the traditional neurotransmission imbalance hypothesis. However, the anticonvulsive pharmacotherapy currently available remains unsatisfactory. To develop new antiepileptic drugs with novel antiepileptic mechanisms, we have tested the antiepileptic actions of ONO-2506, a glial modulating agent, and its effects on tripartite synaptic transmission. EXPERIMENTAL APPROACHDose-dependent effects of ONO-2506 on maximal-electroshock seizure (MES), pentylenetetrazol-induced seizure (PTZ) and epileptic discharge were determined in a genetic model of absence epilepsy in mice (Cacna1a tm2Nobs/tm2Nobs strain). Antiepileptic mechanisms of ONO-2506 were analysed by examining the interaction between ONO-2506 and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on release of L-glutamate, D-serine, GABA and kynurenic acid in the medial-prefrontal cortex (mPFC) of freely moving rats using microdialysis and primary cultured rat astrocytes. KEY RESULTS ONO-2506 inhibited spontaneous epileptic discharges in Cacna1atm2Nobs/tm2Nobs mice without affecting MES or PTZ. Given systemically, ONO-2506 increased basal release of GABA and kynurenic acid in the mPFC through activation of both neuronal and glial exocytosis, but inhibited depolarization-induced releases of all transmitters. ONO-2506 increased basal glial release of kynurenic acid without affecting those of L-glutamate, D-serine or GABA. However, ONO-2506 inhibited AMPA-induced releases of L-glutamate, D-serine, GABA and kynurenic acid. CONCLUSIONS AND IMPLICATIONSONO-2506 did not affect traditional convulsive tests but markedly inhibited epileptic phenomena in the genetic epilepsy mouse model. ONO-2506 enhanced release of inhibitory neuro-and gliotransmitters during the resting stage and inhibited tripartite transmission during the hyperactive stage. The results suggest that ONO-2506 is a novel potential glial-targeting antiepileptic drug. LINKED ARTICLEThis article is commented on by Onat, pp. 1086-1087 of this issue. To view this commentary visit http://dx

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.