The synthesis and properties of a functional fluorescent cholesterol analog

The synthesis of a new fluorescent cholesterol analog is described. The analog contains a cholesterol nucleus attached via a hydrophilic spacer to N-4-nitrobenzo-2-oxa-1,3-diazole. Since the cholesterol moiety is not perturbed this molecule probably interacts with lipid bilayers in much the same way as cholesterol itself does. The compound can be readily incorporated into small unilamellar vesicles by sonicating a mixture of it with egg yolk phosphatidylcholine in a buffer. Furthermore, the analog can be incorporated into preformed membranes either by exchange from vesicles containing the analog or by uptake from sonicated micelles of the analog. Thus this analog shows potential as a useful tool for studying the interactions of cholesterol with cell membranes.     

Inhibitors of retinyl ester formation also prevent the biosynthesis of 11-cis-retinol

Lecithin retinol acyl transferase (LRAT) from the retinyl pigment epithelium is potently inhibited by all-trans-retinyl alpha-bromoacetate in the micromolar range. The inhibition is competitive and reversible. The retinyl pigment epithelium also contains an enzymatic activity capable of converting added all-trans-retinol into 11-cis-retinol. This isomerization is likely to require the intermediate formation of all-trans-retinyl esters, which are themselves produced by LRAT action. Here this possibility is directly tested by studying the effect of all-trans-retinyl alpha-bromoacetate on the isomerization reaction. When pigment epithelium membranes are preincubated with all-trans-retinyl alpha-bromoacetate, they form neither retinyl esters nor 11-cis-retinol from added all-trans-retinol. However, if the pigment epithelium membranes are first allowed to form all-trans-retinyl esters from all-trans-retinol before the addition of all-trans-retinyl alpha-bromoacetate, then 11-cis-retinol formation proceeds at close to the rate found in the absence of inhibitor. In addition, 11-cis-retinyl esters are not formed under these conditions, eliminating the possibility of a direct ester-ester isomerization route. Therefore, all-trans-retinyl esters are obligate intermediates in the biosynthesis of 11-cis-retinol.     

Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes.

Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.     

Altered caveolin-3 expression disrupts PI(3) kinase signaling leading to death of cultured muscle cells

Caveolae and their coat proteins, caveolins, co-ordinate multiple signaling pathways. Caveolin-3 is a muscle-specific caveolin isoform that is deficient in limb girdle muscular dystrophy type 1 C (LGMD1C). Paradoxically, overexpression of this protein also causes muscle degeneration in vivo. We hypothesize that altered membrane expression of caveolin-3 in muscle cells causes a degenerative phenotype by disrupting the co-ordination of signaling pathways that are critical to the maintenance of cell survival. Here, we show for the first time that, in normal muscle cells subjected to oxidative stress, the phosphatidylinositol (3) kinase (PI(3) kinase)-associated proteins PDK1 and Akt associate with caveolae where they bind to caveolin-3, and that normal activation of this pathway promotes cell survival. Either increased or decreased expression of caveolin-3 at the membrane caused an increased susceptibility to oxidative stress, and myotube survival was markedly improved by PI(3) kinase inhibition. This occurred concomitantly with altered phosphorylation of the pro-apoptotic proteins GSK3beta and Bad, despite normal levels of Akt activation. Taken together, our results demonstrate that altered caveolin-3 expression can change the outcome of PI(3) kinase activation from cell survival to cell death. These findings indicate that normal expression and localization of caveolin-3 are required to appropriately co-ordinate PI(3) kinase/Akt-mediated cell survival signaling, and suggest that this pathway may be an effective therapeutic target for the treatment of muscular dystrophies associated with caveolin-3 mutations.     

Small molecule RPE65 antagonists limit the visual cycle and prevent lipofuscin formation

The accumulation of the lipofuscin fluorophores in retinal pigment epithelial (RPE) cells leads to the blinding degeneration characteristic of Stargardt disease and related forms of macular degeneration. RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cycle. Inhibiting visual cycle function with small molecules is required to prevent the formation of the retinotoxic lipofuscins. This in turn requires identification of rate-limiting steps in the operation of the visual cycle. Specific, non-retinoid isoprenoid compounds are described here, and shown through in both in vitro and in vivo experiments, to serve as antagonists of RPE65, a protein that is essential for the operation of the visual cycle. These RPE65 antagonists block regeneration of 11-cis-retinal, the chromophore of rhodopsin, thereby demonstrating that RPE65 is at least partly rate-limiting in the visual cycle. Furthermore, chronic treatment of a mouse model of Stargardt disease with the RPE65 antagonists abolishes the formation of A2E. Thus, RPE65 is also on the rate-limiting pathway to A2E formation. These nontoxic isoprenoid RPE65 antagonists are candidates for the treatment of forms of macular degeneration wherein lipofuscin accumulation is an important risk factor. These antagonists will also be used to probe the molecular function of RPE65 in vision.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.