Faculty Bibliography

The biological functions of vitamin A in the epidermis are mediated by all-trans retinoic acid, which is biosynthesized from retinol in two oxidative reactions. The first step involves enzymatic conversion of retinol to retinaldehyde. The physiological significance and relative contributions of the various retinol dehydrogenases to the oxidation of retinol in epidermal cells remain unclear. We report the characterization of a retinol dehydrogenase/reductase of the SDR superfamily, hRoDH-E2, which is abundantly expressed in the epidermis, epidermal appendages and in cultured epidermal keratinocytes. Both in live keratinocytes and in isolated keratinocyte microsomes, where the enzyme normally localizes, hRoDH-E2 functions as a bona fide retinol dehydrogenase. In the prevailing oxidative reaction it recognizes both free- and CRBP-bound retinol, and shows preference toward NADP as a co-substrate. In comparison, hRoDH-E2 retinol dehydrogenase activity in the simple epithelial HEK 293 cells is much lower and in CHO cells is non-existent. hRoDH-E2 transcripts are distributed throughout the epidermal layers but are more abundant in the basal cells. In contrast, the protein is detected predominantly in the basal and the most differentiated living layers. Its synthesis is negatively regulated by retinoic acid. The biochemical properties and the differential expression of hRoDH-E2 in the strata where retinoic acid signaling is critical for epidermal homeostasis support a conclusion that hRoDH-E2 bears the characteristics of the major microsomal retinol dehydrogenase activity in the epidermal keratinocytes in physiological circumstances.

BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) promotes excitotoxic and ischemic injury within the brain. These findings have implications for the use of tPA in the treatment of acute ischemic stroke. The plasminogen activator from vampire bat (Desmodus rotundus) saliva (D rotundus salivary plasminogen activator [DSPA]; desmoteplase) is an effective plasminogen activator but, in contrast to tPA, is nearly inactive in the absence of a fibrin cofactor. The purpose of this study was to compare the ability of DSPA and tPA to promote kainate- and N-methyl-D-aspartate (NMDA)-induced neurodegeneration in tPA-/- mice and wild-type mice, respectively. METHODS: tPA-/- mice were infused intracerebrally with either tPA or DSPA. The degree of neuronal survival after hippocampal injection of kainate was assessed histochemically. Wild-type mice were used to assess the extent of neuronal damage after intrastriatal injection of NMDA in the presence of tPA or DSPA. Immunohistochemistry and fibrin zymography were used to evaluate DSPA and tPA antigen or activity. RESULTS: Infusion of tPA into tPA-/- mice restored sensitivity to kainate-mediated neurotoxicity and activation of microglia. DSPA was incapable of conferring sensitivity to kainate treatment, even when infused at 10-fold higher molar concentration than tPA. The presence of tPA also increased the lesion volume induced by NMDA injection into the striatum of wild-type mice, whereas DSPA had no effect. CONCLUSIONS: DSPA does not promote kainate- or NMDA-mediated neurotoxicity in vivo. These results provide significant impetus to evaluate DSPA in patients with ischemic stroke.

PURPOSE: The functional characteristics of tight junctions in the outer blood-retinal barrier change during embryonic development and in the presence of disease. A culture model of developing retinal pigment epithelium (RPE) was used to examine the regulation of the tight junctions. METHODS: RPE from chick embryos was cultured on filters that separated the apical and basal medium compartments. Cultures were maintained in various combinations of serum-free medium, serum-free medium that was conditioned by neural retinas, or serum-free medium that was supplemented with bovine pituitary extract, serum, or various hormones. Function was monitored by the transepithelial electrical resistance (TER) or the permeation of small organic tracers. Structure was monitored by immunofluorescence and freeze-fracture electron microscopy. RESULTS: Functional analysis indicated differences in permeability among RPE of different embryonic age and culture conditions. In serum-free medium, the tight junctions were leaky or failed to form. Barrier properties increased if pituitary extract was added to the basal medium chamber or retina-conditioned medium was added to the apical chamber. Retina-conditioned medium was more effective at organizing tight junctional strands into a continuous network, but bovine pituitary extract appeared to modulate the permeability of that network. In combination, they synergistically elevated the TER to physiological levels. Although the thyroid hormone T3 had no effect, serum in the apical medium chamber inhibited the ability of RPE cells to respond to retina-conditioned medium. CONCLUSIONS: Diffusible factors secreted by the neural retina acted synergistically with basolateral stimulation to regulate the structure and function of RPE tight junctions. Serum on the apical side of the RPE monolayer inhibited the ability of retinal factors to upregulate the tight junction barrier.

Latent transforming growth factor beta-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor beta to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed

Regulation of stem cell division is of particular interest, both for studies of development and for stem cell therapeutics. In this issue of Neuron, Bello et al. show that the number of divisions of Drosophila neural stem cells is limited, in a region-specific manner, by regulated apoptosis in response to a pulse of expression of the Hox gene abdominal-A (abdA).

Germ cells preserve an individual's genetic information and transmit it to the next generation. Early in development germ cells are set aside and undergo a specialized developmental programme, a hallmark of which is the migration from their site of origin to the future gonad. In Drosophila, several factors have been identified that control germ-cell migration to their target tissues; however, the germ-cell chemoattractant or its receptor have remained unknown. Here we apply genetics and in vivo imaging to show that odysseus, a zebrafish homologue of the G-protein-coupled chemokine receptor Cxcr4, is required specifically in germ cells for their chemotaxis. odysseus mutant germ cells are able to activate the migratory programme, but fail to undergo directed migration towards their target tissue, resulting in randomly dispersed germ cells. SDF-1, the presumptive cognate ligand for Cxcr4, shows a similar loss-of-function phenotype and can recruit germ cells to ectopic sites in the embryo, thus identifying a vertebrate ligand-receptor pair guiding migratory germ cells at all stages of migration towards their target

TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective