Development of neuronal locus specificity in Xenopus retinal ganglion cells after surgical eye transection after fusion of whole eyes

A developmental program is established in the stage 28–32 optic cup of Xenopus embryos, which specifies the permanent AP and DV reference axes for positional information in the retina, and thereby determines the pattern of spatial deployment of ganglion cell locus specificities subserving assembly of retinotopically organized connections in the tectum. This developmental program has previously proved unmodifiable in intact eye primordia submitted to a variety of rotation, transplantation, and tissue culture conditions. Here we report that the program can be modified by surgical transection of stage 32 eye primordia (with subsequent fusion of the disconnected halves to reconstitute a whole eye) and by fusion of whole stage 38 eyes, although most of the transected eyes did develop normal visuotectal projections. The remaining vertically transected eyes, and all eyes formed when a left and right stage 38 eye fused along apposed temporal edges, developed “double-nasal compound” projections to the tectum: the nasal and temporal halves of the adult retina each projected to the entire tectum, and each tectal locus was driven from two stimulus positions symmetrically disposed about the vertical meridian. The remaining horizontally transected eyes, and all eyes formed when a left and right stage 38 eye fused along apposed dorsal edges, developed “double-ventral compound” projections to the tectum: the dorsal and ventral halves of the adult retina each projected to the entire tectum, and each tectal locus was driven from two stimulus positions symmetrically disposed about the horizontal meridian. The results are considered in terms of (1) the kinds of cellular processes that could mediate the observed modifications in the original developmental program; (2) the nature and stability of the program; and (3) the general suitability of eye fragment-fusion experiments for analysis of the assembly of retinotectal connections.     

The introduction of the C-22–C-23 ethylenic linkage in ergosterol biosynthesis

Methods for the chemical synthesis of [23-(3)H(2)]lanosterol, [23,25-(3)H(3)]24-methyldihydrolanosterol and [24,28-(3)H(2)]24-methyldihydrolanosterol are described. It is shown that, in the biosynthesis of ergosterol from [26,27-(14)C(2),23-(3)H(2)]lanosterol by the whole cells of Saccharomyces cerevisiae, one of the original C-23 hydrogen atoms is lost and the other is retained at C-23 of ergosterol. It is also shown that 24-methyldihydrolanosterol is converted into ergosterol in good yield and without prior conversion into a 24-methylene derivative. On the basis of these results possible pathways for the formation of the ergosterol side chain from a 24-methylene side chain are discussed.     

Avidin-like domain in an epidermal growth factor homolog from a sea urchin

We have found that a protein from the purple sea urchin has a carboxyl-terminal domain with striking sequence similarity to chicken avidin and bacterial streptavidin. All our evidence supports the homology of these sequences. Tetramers of avidin and streptavidin bind biotin strongly; the biotin binding site involves two to four tryptophans and probably an adjacent lysine in each chain. The presence of four tryptophans at equivalent positions in the sea urchin protein domain suggests that it may also be able to bind biotin and inhibit cell growth, as do the two other proteins. Alternatively, this domain may have acquired a new role as part of a multidomain protein.     

Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro.

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.     

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.