Expression and localization of CERKL in the mammalian retina, its response to light-stress, and relationship with NeuroD1 gene.

Mutations in the Ceramide kinase like (CERKL) gene are associated with retinitis pigmentosa (RP26) and cone-rod dystrophy. CERKL is homologous to Ceramide kinase (CERK), and its function is still unknown. The purpose of this study was to test the expression and distribution of this gene and its protein in rat and in mouse tissues, in light-stressed rat retinas and in the retinas of NeuroD1 knock-out mice to understand the role of CERKL in the retina. Expression of Cerkl and Cerk mRNA was determined by quantitative RT-PCR. Expression of the protein was determined by Western blotting with anti-CERKL antibody. Localization of the protein was determined by using immunofluorescence microscopy. With qRT-PCR, we revealed that the relative mRNA expression of Cerkl was the highest in the retina among all the rat tissue tested; it was >10-fold higher than in the brain. On the other hand, Cerk has ubiquitous expression and its relative abundance is >2 fold of Cerkl in the retina. Cerkl was expressed minimally in the developing mouse eyes and reached a peak at retinal maturity at 2 months. Western blots of retinal tissues revealed two major CERKL protein bands: 59 kDa (C1) and 37 kDa (C2). However, only C2 CERKL was found in the rat retinal rod outer segment (ROS) at level of that was not changed in light vs. dark adaptation. In the light-stressed retina, expression of Cerkl mRNA increased significantly, which was reflected in only on C2 CERKL protein. The CERKL protein localized prominently to the ganglion cells, inner nuclear layers (INL), retinal pigment epithelial (RPE) cells, and photoreceptor inner segments in the retinal sections. Nuclear localization of CERKL was not affected in RPE, INL and the ganglion cell layers in the light-stressed retina; however, the perinuclear and outer segment locations appear to be altered. In the NeuroD1 knock-out mouse retina, the expression of Cerkl mRNA and protein decreased and that decrease also pertains to C2 CERKL. In conclusion, the retina had the highest level of Cerkl mRNA and protein expression, which reached its maximum in the adult retina; CERKL localized to ROS and RPE cells and the light-adaptation did not change the level of CERKL in ROS; light-stress induced Cerkl expression in the retina; and its expression decreased in NeuroD1 knock-out retina. Thus, CERKL may be important for the stress responses and protection of photoreceptor cells.

Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of CaCo 2 colon cancer cell line in vitro.

Calycopteris floribunda Lam., commonly known as 'goichia lata or goache lata', is a large climbing woody shrub from Bangladesh, and well distributed in a number of other south-east Asian countries. Traditionally, C. floribunda has been used in colic, as an antihelminthic, astringent and carminative, and for the treatment of diarrhoea, dysentery, jaundice and malaria in many countries including Bangladesh. Pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) has been isolated from the leaves of C. floribunda by repeated column chromatography on silica gel, and the structure confirmed by spectroscopic means. While the general toxicity of pachypodol was determined by the brine shrimp lethality assay, the cytotoxic potential of this flavonoid has been evaluated by the Promega's CellTiter 96 Non-Radioactive Cell Proliferation Assay using the CaCo-2 colon cancer cell line (IC(50) = 185.6 microM). A summary of the biological activities of pachypodol reported to date is also presented.