Alexander N. Gubin

Prospective identification of erythroid elements in cultured peripheral blood.

We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48- cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48- cells increased to 34.7 + 14.9% (p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcellulose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.

Origin and Evolution of Retinoid Isomerization Machinery in Vertebrate Visual Cycle: Hint from Jawless Vertebrates

In order to maintain visual sensitivity at all light levels, the vertebrate eye possesses a mechanism to regenerate the visual pigment chromophore 11-cis retinal in the dark enzymatically, unlike in all other taxa, which rely on photoisomerization. This mechanism is termed the visual cycle and is localized to the retinal pigment epithelium (RPE), a support layer of the neural retina. Speculation has long revolved around whether more primitive chordates, such as tunicates and cephalochordates, anticipated this feature. The two key enzymes of the visual cycle are RPE65, the visual cycle all-trans retinyl ester isomerohydrolase, and lecithin:retinol acyltransferase (LRAT), which generates RPE65’s substrate. We hypothesized that the origin of the vertebrate visual cycle is directly connected to an ancestral carotenoid oxygenase acquiring a new retinyl ester isomerohydrolase function. Our phylogenetic analyses of the RPE65/BCMO and N1pC/P60 (LRAT) superfamilies show that neither RPE65 nor LRAT orthologs occur in tunicates (Ciona) or cephalochordates (Branchiostoma), but occur in Petromyzon marinus (Sea Lamprey), a jawless vertebrate. The closest homologs to RPE65 in Ciona and Branchiostoma lacked predicted functionally diverged residues found in all authentic RPE65s, but lamprey RPE65 contained all of them. We cloned RPE65 and LRATb cDNAs from lamprey RPE and demonstrated appropriate enzymatic activities. We show that Ciona ß-carotene monooxygenase a (BCMOa) (previously annotated as an RPE65) has carotenoid oxygenase cleavage activity but not RPE65 activity. We verified the presence of RPE65 in lamprey RPE by immunofluorescence microscopy, immunoblot and mass spectrometry. On the basis of these data we conclude that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerohydrolase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates.

The Mechanism of Fenretinide (4-HPR) Inhibition of β-carotene Monooxygenase 1. New Suspect for the Visual Side Effects of Fenretinide

Fenretinide (4-hydroxy(phenyl)retinamide; 4-HPR), a synthetic retinoid derivative, shows anticancer potential in clinical trials for treatment of breast cancer. Common side effects associated with fenretinide treatment include night blindness initially attributed to inhibition of RBP4 (retinol-binding protein 4)-binding to retinol and consequent impaired retinol transport. However, fenretinide affects RBP4−/− animals similarly to wild-type mice, and thus, the reason for the effects of fenretinide remains elusive. It has been suggested that ®-carotene monooxygenase 1 (BCMO1) might supply all-trans retinal as an accessory source of vitamin A for the visual cycle. We found that fenretinide is a strong inhibitor of mouse BCMO1 (K i ∼ 1.2 μM), acting noncompetitively. In contrast, other retinoids, such as retinyl palmitate and retinyl acetate, as well as other biologically active aromatic compounds (capsaicin and resveratrol, and the amino analog of fenretinide) do not substantially inhibit BCMO1 activity. To study the mechanism of inhibition, we deleted portion of an interstrand loop of BCMO1 (metazoan loop) to generate BCMO1⊗336-345. This mutant had impaired enzymatic activity, but was not substantially inhibited by fenretinide. Thus, we demonstrate that fenretinide is a strong noncompetitive inhibitor of BCMO1 and that the metazoan loop influences binding of this retinoid. Our data point to an additional mechanism of fenretinide-induced night blindness through inhibition of BCMO1.