AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs.

Interstrand cross-links at T(A/T)4A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T)4A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200-1000 base pairs of approximately 85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (approximately 4.7-7.1 lesions/kilobase pair/microM drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (approximately 0.8-1.3 and approximately 0.9 lesions/kilobase pair/microM drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like AT-rich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.

Retinyl esters in the vertebrate neuroretina.

High-performance liquid chromatography (HPLC) was employed to measure retinyl esters in the vertebrate retina. Both retina and retinal pigment epithelium (RPE) from frog, chicken, and bovine eyes were studied. In comparison to the RPE, the retina possessed a significant level of 11-cis and all trans retinyl palmitate. Using a sensitive radioassay, we also detected the presence of retinyl ester hydrolase (REH) activity in homogenates prepared from both retina and RPE. The rate of retinyl ester hydrolysis in these retinas was sufficiently high to supply retinal chromophores for the metabolic renewal and for the regeneration of visual pigments. In comparison to retinyl esters in the RPE, retinyl esters in the retina are located much closer to the sites of visual pigment synthesis and regeneration. Hence it is possible that these retinyl esters play a more important role in the visual cycle than those in the RPE.

Visual pigments and retinoids in the Mongolian jird.

The visual cells, visual pigments and major retinoids of the Mongolian jird (Meriones unguiculatus) were examined. Light and electron microscope analyses show that these jirds had mainly rod photoreceptors. Octylglucoside extracts prepared from their retinas contained only rhodopsin with a maximum absorption at 497 nm and a concentration of 0.51 nmol per retina. Employing a standard method of high performance liquid chromatography (HPLC), the pigment epithelium from each eye was found to possess 0.52 nmol of retinyl palmitate (the most abundant form of retinyl ester) along with a small amount of retinol (0.02 nmol). Most of the retinoids in the body of these animals are stored in the liver, in the form of retinyl palmitate (1228.80 nmol per gram liver). As the Mongolian jird is small, inexpensive and readily available, this animal is a mammalian species suitable for the research of the biochemistry of retinoids and vision.

Effects of glycosylation inhibitors on the frog retina.

Effects of the intraocular injection of three inhibitors of glycosylation (tunicamycin, castanospermine, and swainsonine) on the rhodopsin content and the integrity of disc membranes in frog retina were studied. The administration of 10 or 100 micrograms of tunicamycin resulted in a 78% loss of rhodopsin in the frog retina which also exhibited a significant reduction in the length of photoreceptor outer segments (as examined under light microscope). This suggests that the synthesis and/or insertion of rhodopsin into the disc membrane is inhibited by tunicamycin. In contrast, injections of up to 250 micrograms of castanospermine and swainsonine resulted in neither a decrease in rhodopsin content nor a change in the length of photoreceptor outer segments. Examination of retinal tissue homogenates for alpha Mannosidase and alpha-Glucosidase activities revealed homogenates for alpha Mannosidase and alpha-Glucosidase activities revealed these hydrolase activities to be significantly decreased (70%). We suggest the possibility that specific oligosaccharide processing reactions may not be required for the insertion of rhodopsin and subsequent assembly of disc membranes in frog photoreceptors.