Modification of low density lipoprotein (LDL) by free radical oidat renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo In atherosclerotic lesions and in the plasma of diabetic i , suggesting that Upoprotein-induced tissue damage may occur in cen diseases. We undertook purifiation and Identicati of the major cytotoxin In oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance Identified the purified toxin as 7p-hydroperoxycholest-5-en-3I3-ol Vascular cells are susceptible to the toxic effects of low density lipoprotein (LDL) or very low density lipoprotein (VLDL) after modification of the lipoproteins by free radical oxidation (1-4). Oxidation of LDL leads to cytotoxin formation whether oxidation is mediated by lipoxygenases (5), metal ions (6), or ultraviolet irradiation (7) in cell-free systems or by the action of free radicals from cultured endothelial cells (8), vascular smooth muscle cells (8), neutrophils (9), or stimulated human monocytes (10). The cytotoxic moiety of oxidized LDL (oxLDL) is extractable with organic solvents (4), and numerous candidate substances could be proposed to explain its toxic action (11).Oxidized lipoproteins occur in vivo in vascular lesions (12)(13)(14) and in plasma of certain diabetic subjects (15). OxLDL has been proposed to play a causal role in atherosclerosis (11,(16)(17)(18) is an important step toward testing evolving theories of atherogenesis that include lipoprotein oxidation (11,(16)(17)(18) and vascular injury (20,21) as putative early events.by dialysis against 2-6 /uM cupric sulfate for various times (23). OxLDL preparations were dialyzed against 0.15 M NaCl/0.5 mM EDTA, pH 8.5, to remove cupric ions. Relative oxidation, measured as thiobarbituric acid reactivity (6,24), was equivalent to 4-6 nmol of malondialdehyde (MDA) per mg of LDL cholesterol.Human foreskin fibroblasts were plated in a 1:1 (vol/vol) mixture of Dulbecco's minimal essential medium and Ham's F-12 medium (DME/F-12) supplemented with 5% (vol/vol) fetal bovine serum (6) Aliquots of5 mg ofnative LDL or oxLDL were lyophilized overnight, and the lipid was extracted with 5 ml of acetone. The mixture was sonicated for 10-20 sec, mixed for 1 min, and allowed to stand for 20 min. After centrifugation at 1000x g for 10-20 min, the residue was reextracted twice, as above. Preliminary experiments revealed that the recovery of the cytotoxic activity by this extraction procedure was equivalent or superior to other means, including chloroform/ methanol extraction ofaqueous lipoprotein solutions. Pooled extracts were dried under nitrogen and redissolved in isopropanol/acetonitrile, 1:1 (vol/vol). After centrifugation for 5 min at 1000 x g, the supernatant was analyzed by reversephase HPLC (Waters 1LBondapak C1s preparative column).The solvent gradient for elution consisted of water/ acetonitrile, 1:1 (vol/vol), which was increased over 5 ...

An in situ method for generating Et2BOCH3 from triethylborane and methanol without using any other catalysts is described. Using the Et2BOCH3 thus generated as a chelating agent, syn 1,3-diols are prepared in ≥ 98% stereochemical purity by reducing β-hydroxyketones with sodium borohydride.

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a oneway reaction that activates transducin (G t ) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C 11 =C 12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged G t activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates G t in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.is the visual pigment found in rod outer segments of vertebrate and invertebrate photoreceptors that mediates the transformation of light into vision (1-5). By contrast, microbial Rhs mediate both the energy conversion and cell signaling required for cell survival (2, 6, 7). All classes of Rhs feature a heptahelical transmembrane structure that incorporates a covalently bound retinal chromophore, but they differ in their ability to interconvert between their two spectral absorption states driven by light exposure (2). Although vertebrate Rh undergoes a one-way photobleaching reaction of the retinal chromophore after light absorption (8), microbial Rhs exhibit an intrinsic photocyclic behavior with no chromophore release (2,8). This makes vertebrate Rhs unsuitable for optogenetic applications that require reversible control over effector ligands. Nevertheless, all Rhs require a cis-trans/trans-cis isomerization of their chromophores to trigger a protein conformational change that mediates the downstream signaling cascade or energy conversion (2,8). Previous studies on bovine Rh regenerated with sixcarbon-ring retinal chromophores (Rh6mr) featuring a locked C 11 =C 12 cis-trans isomerization (SI Appendix, Fig. S1) have implied their ability to activate the G protein transducin (G t ). These results suggest an alternative mechanism that can propagate the downstream visual cascades (9)(10)(11)(12)(13)(14). In this study, we provide direct evidence that Rh6mr can activate G t by an atypical cis-to-dicis isomerization that is also photocyclic, undergoing an 11,13-dicis-to-11-cis reisomerization. Even though it is believed that cis-trans isomerization is required to achieve the conformational change in opsin needed for G t activation, our results generalize this prerequisite to any isomerization that can achieve the same con...

4-Aminothiazolyl analogues of the antibacterial natural product GE2270 A (1) were designed, synthesized, and evaluated for gram positive bacteria growth inhibition. The aminothiazole-based chemical template was evaluated for chemical stability, and its decomposition revealed a novel, structurally simplified, des-thiazole analogue of 1. Subsequent stabilization of the 4-aminothiazolyl functional motif was achieved and initial structure activity relationships defined.

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