Novel TEMPO-PEG-RGDs conjugates remediate tissue damage induced by acute limb ischemia/reperfusion.

We have recently developed new Tempo-PEG-RGDs conjugates and have quantitatively examined their antithrombotic and antioxidant capabilities. These compounds were therapeutically beneficial when characterized in both in vitro platelet aggregation assays and a rat model of arterial thrombosis. Moreover, these compounds demonstrated significant protection from organ damage in a rat model of ischemia/reperfusion. Our data indicate that Tempo-PEG-RGDs represent a new class of adjuvants with therapeutic efficacy in acute and transient ischemic damage.

New rhodamine nitroxide based fluorescent probes for intracellular hydroxyl radical identification in living cells.

The synthesis, characteristics, and biological applications of a series of new rhodamine nitroxide fluorescent probes that enable imaging of hydroxyl radicals (•OH) in living cells are described. These probes are highly selective for •OH in aqueous solution, avoiding interference from other reactive oxygen species (ROS), and they facilitate •OH imaging in biologically active samples. The robust nature of these probes (high specificity and selectivity, and facile synthesis) offer distinct advantages over previous methods for •OH detection.

Mechanisms involved in A2E oxidation.

A2E is one of the bis-retinoid pyridinium compounds that accumulate as lipofuscin pigments in retinal pigment epithelial (RPE) cells in association with aging and in some inherited forms of retinal degeneration. Here we observed that 430nm irradiation of A2E in the presence of the spin trap DMPO, led to the appearance of a superoxide dismutase-inhibitable electron paramagnetic resonance (EPR) spectrum characteristic of DMPO-OH; this finding was indicative of hydroxyl radical (OH) formation following initial spin trapping of superoxide anion by DMPO. We also observed an increase in dihydroethidium (HEt) fluorescence and luminol-based chemiluminescence that on the basis of inhibition by superoxide dismutase, was indicative of superoxide anion generation when A2E was irradiated at 430nm in cell-free systems. Nevertheless, while A2E was readily oxidized in the presence of a singlet oxygen generator, superoxide anion did not serve to oxidize A2E. Specifically, by HPLC quantitation and FAB-mass spectroscopy, there was no evidence of A2E oxidation when A2E was incubated with a superoxide anion generator (xanthine/xanthine oxidase) in a variety of solvents (100% PBS, 30% DMSO in PBS, 100% MeOH and CHCl3) or in the presence of detergent. On the other hand, however, peroxy-A2E, an oxidized form of A2E with an endoperoxide moiety on the short-arm of the molecule, readily underwent further oxygen addition when incubated with xanthine/xanthine oxidase. Superoxide anion may be generated by irradiation of A2E but is not involved in the early events that oxidize A2E. Superoxide can contribute to the further oxidation of already-oxidized A2E.

Immunochemical recognition of A2E, a pigment in the lipofuscin of retinal pigment epithelial cells.

The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with age and is particularly abundant in some retinal disorders. To generate a polyclonal antibody that recognizes this pyridinium bisretinoid molecule, we immunized rabbits with bovine serum albumin (BSA) conjugates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety. Analysis by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it a suitable antigen for immunization. By immunocytochemical staining, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various retinoids. Preimmune serum was nonreactive. In fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and by a blue-shift in the emission maximum consistent with a change in A2E milieu upon antibody binding. The changes in fluorescence emission upon antibody binding could reflect several processes including restrictions on trans-cis isomerization and intersystem crossing of photo-excited A2E.

The effect of ultraviolet-depleted light on the flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea.

An early investigation at the Biosphere-2 Laboratory, an artificial ecosystem in the Arizona desert, had shown that the flavonoid content of cacti grown in glass-filtered solar light was lower than of cacti grown in normal solar light. This was attributed to the absence of ultraviolet (UV) radiation, which is required for flavonoid biosynthesis. In this study, two species of Opuntia cacti were grown in solar and UV-depleted light, and their flavonol contents of different tissues were determined by HPLC. O. wilcoxii, previously raised in the absence of UV light, was exposed to normal solar light. The flavonol content of young O. wilcoxii pads was 28-fold higher when grown in solar light as compared to UV-depleted light. The flavonol contents of mature outer tissues were only slightly higher. O. violacea, previously raised in solar light, was also maintained in the same UV-depleted artificial ecosystem. The flavonol content after hydrolysis of outer tissues was similar, whether grown in solar light or UV-depleted light. We attribute these responses to different biosynthetic and metabolic rates of young vs. mature plant tissues; slow-growing mature tissues neither produce nor metabolize compounds as quickly as immature tissues. These findings indicate that artificial ecosystems can influence the production of natural products in cultivated plants.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus.

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.

Photooxidation of A2-PE, a photoreceptor outer segment fluorophore, and protection by lutein and zeaxanthin.

A2-PE is a pigment that forms as a byproduct of the visual cycle, its synthesis from all-trans-retinal and phosphatidylethanolamine occurring in photoreceptor outer segments. A2-PE is deposited in retinal pigment epithelial (RPE) cells secondary to phagocytosis of shed outer segment membrane and it undergoes hydrolysis to generate the RPE lipofuscin fluorophores, A2E, iso-A2E and other minor cis-isomers of A2E. We have demonstrated that A2-PE can initiate photochemical processes that involve the oxidation of A2-PE and that, by analogy with A2E are likely to include the formation of reactive moieties. We also show that potential sources of protection against the photooxidation of A2-PE are the lipid-soluble carotenoids zeaxanthin and lutein (xanthophylls), that constitute the yellow pigment of the macula. Irradiation of A2-PE in the presence of lutein or zeaxanthin suppressed A2-PE photooxidation and in experiments in which we compared the antioxidant capability of zeaxanthin and lutein to alpha-tocopherol, the carotenoids were more potent. Additionally, the effect with zeaxanthin was consistently more robust than with lutein and when alpha-tocopherol was combined with either carotenoid, the outcome was additive. Lutein, zeaxanthin and alpha-tocopherol were all efficient quenchers of singlet oxygen. We have also shown that lutein and zeaxanthin can protect against A2-PE/A2E photooxidation without appreciable consumption of the carotenoid by chemical reaction. This observation contrasts with the pronounced susceptibility of A2E and A2-PE to photooxidation and is of interest since lutein, zeaxanthin, A2E and A2-PE all have conjugated systems of carbon-carbon double bonds terminating in cyclohexenyl end-groups. The structural features responsible for the differences in quenching mechanisms are discussed. It has long been suspected that macular pigment protects the retina both by filtering high-energy blue light and by serving an antioxidant function. Evidence presented here suggests that the photochemical reactions against which lutein and zeaxanthin protect, may include those initiated by the A2-PE. Quantitative HPLC analysis revealed that in eyecups of C57BL/6J and BALB/cByJ mice, levels of A2-PE were several fold greater than the cleavage product, A2E. Taken together, these results may have implications with respect to the involvement of A2-PE formation in mechanisms underlying blue light-induced photoreceptor cell damage and may be significant to retinal degenerative disorders, such as those associated with ABCA4 mutations, wherein there is a propensity for increased A2-PE synthesis.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu–Asn–Leu–Lys–Gly–Ile–Phe–Lys–Lys–Val–Ala–Ser–Leu–Leu–Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear á-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic á-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of á-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.

Retinoylserine and retinoylalanine, natural products of the moth Trichoplusia ni.

Insect cells convert vitamin A into a number of retinoids that are evolutionarily conserved with those of mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic acid peptides, N-trans-retinoylserine (1) and N-trans-retinoylalanine (2), have been isolated from a cell line of the common cabbage looper, Trichoplusia ni. These are the first examples of naturally occurring retinoic acid linked to amino acids through an amide bond; the amino acid moieties are depicted in the more common l-configuration, although the absolute configuration was not determined due to the minuscule sample amount.

Characterization of peroxy-A2E and furan-A2E photooxidation products and detection in human and mouse retinal pigment epithelial cell lipofuscin.

The nondegradable pigments that accumulate in retinal pigment epithelial (RPE) cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease, a blindness macular disorder of juvenile onset. This autofluorescent material may also contribute to the etiology of age-related macular degeneration. The best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side arms extending from a pyridinium ring. Evidence indicates that photochemical mechanisms initiated by excitation from the blue region of the spectrum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates. By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or two oxygens at a time, together with structural analysis by heteronuclear single quantum correlation-NMR spectroscopy, we demonstrated that the oxygen-containing moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monoperoxide. We have shown that the oxidation sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on the shorter arm. By liquid chromatography-mass spectrometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr-/- mice, a model of Stargardt disease. Because the cytotoxicity of endoperoxide moieties is well known, the production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage ensuing from A2E photooxidation.

Three challenges toward the assignment of absolute configuration of gymnocin-B.

The absolute configuration of gymnocin-B has been determined to be (S)-10 and (S)-37. Three challenges toward the configurational assignment of this largest of the polyether marine toxin include (i) introduction of p-(meso-triphenylporphyrin)-cinnamate group (TPPcinnamate) on sterically hindered 10-, 37-hydroxyls under mild conditions, (ii) conformational analysis in the presence of TPPcinnamates at C-10 and C-37 positions on the flexible seven-membered rings embodied in a large polyether ladder-like scaffold structure, and (iii) determination of the chirality at C-10 and C-37 on the basis of porphyrin/porphyrin circular dichroism exciton-coupled interaction over a large distance. The experimentally obtained positive exciton couplet by CD and FDCD of the bis-TPPcin derivative of gymnocin-B is in good agreement with that of theoretically calculated CD of the MMFF optimized structures, by employing DeVoe's coupled oscillator approach, thus establishing the full absolute configuration of gymnocin-B.

Absolute configuration of a hydroxyfuranoid acid from the pelage of the genus Bos, 18-(6S,9R,10R)-bovidic acid.

Pelage extracts of the banteng (Bos javanicus), the domestic cattle (B. taurus), the gaur (B. frontalis), and the yak (B. grunniens) were investigated by FABMS and NMR. An 18-carbon hydroxyfuranoid acid, 1 (10-hydroxy-6,9-oxidooctadecanoic acid), first reported from the wool of the domestic sheep (Ovis aries) was confirmed in B. frontalis; we suspend judgment on the occurrence of this compound in the other species we examined. The stereochemistry of 1, determined by Mosher NMR and CD tweezer methods, was assigned as 6S, 9R, 10R. We propose the name bovidic acid for this and homologous alpha-hydroxylated 2,5-tetrahydrofuranoid carboxylic acids, in reference to the family Bovidae, which represents their sole known natural source.

Photochemistry of A1E, a retinoid with a conjugated pyridinium moiety: competition between pericyclic photooxygenation and pericyclization.

The photochemistry of the retinoid analogue A1E shows an oxygen and solvent dependence. Irradiation of A1E with visible light (lambda(irr) = 425 nm) in methanol solutions resulted in pericyclization to form pyridinium terpenoids. Although the quantum yield for this cyclization is low (approximately 10(-4)), nevertheless the photochemical transformation occurs with quantitative chemical yield with remarkable chemoselectivity and diastereoselectivity. Conversely, irradiation of A1E under the same irradiation conditions in air-saturated carbon tetrachloride or deuterated chloroform produced a cyclic 5,8-peroxide as the major product. Deuterium solvent effects, experiments utilizing endoperoxide, phosphorescence, and chemiluminescence quenching studies strongly support the involvement of singlet oxygen in the endoperoxide formation. It is proposed that, upon irradiation, in the presence of oxygen, A1E acts as a sensitizer for generation of singlet oxygen from triplet oxygen present in the solution; the singlet oxygen produced reacts with A1E to produce cyclic peroxide. Thus, the photochemistry of A1E is characterized by two competing reactions, cyclization and peroxide formation. The dominant reaction is determined by the concentration of oxygen, the concentration of A1E, and the lifetime of singlet oxygen in the solvent employed. If the lifetime of singlet oxygen in a given solvent is long enough, then oxidation (peroxide formation) is the major reaction. If the singlet oxygen produced is quenched by the protonated solvent molecules faster than singlet oxygen reacts with A1E, then cyclization dominates.

A2E, a byproduct of the visual cycle.

A substantial portion of the lipofuscin that accumulates with age and in some retinal disorders in retinal pigment epithelial (RPE) cells, forms as a consequence of light-related vitamin A recycling. Major constituents of RPE lipofuscin are the di-retinal conjugate A2E and its photoisomers. That the accretion of A2E has consequences for the cell, with the adverse effects of A2E being attributable to its amphiphilic structure and its photoreactivity, is consistent with evidence of an association between atrophic age-related macular degeneration (AMD) and excessive lipofuscin accumulation.

A2-rhodopsin: a new fluorophore isolated from photoreceptor outer segments.

A2E and iso-A2E are fluorescent amphiphilic pyridinium bisretinoids involved in age-related macular degeneration (AMD). It is now shown that the presence of high exogenous concentrations of all-trans-retinal in photoreceptor outer segments leads to the formation of A2-rhodopsin (A2-Rh), an unprecedented fluorescent rhodopsin adduct which consists of bisretinoids (A2) linked to each of three lysine residues in rhodopsin (Rh) and which exhibits an emission spectrum similar to A2E. The fluorophore to protein ratio was determined by MALDI-TOF-MS and UV-VIS spectroscopy. Enzymatic degradation with thermolysin and cathepsin D showed that two of the A2 moieties were located in the region of the third cytoplasmic loop and 8th helix of Rh. Examination of A2-Rh and A2-PE (the precursor of A2E) fluorescence in relation to all-trans-retinal concentration indicated that whereas A2-PE formation is favored over that of A2-Rh, for a single rhodopsin molecule only one phosphatidylethanolamine molecule is available to react with all-trans-retinal; this phosphatidylethanolamine is probably tightly associated with the protein.

A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation.

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.

"Venom" of the slow loris: sequence similarity of prosimian skin gland protein and Fel d 1 cat allergen.

Bites inflicted on humans by the slow loris (Nycticebus coucang), a prosimian from Indonesia, are painful and elicit anaphylaxis. Toxins from N. coucang are thought to originate in the brachial organ, a naked, gland-laden area of skin situated on the flexor surface of the arm that is licked during grooming. We isolated a major component of the brachial organ secretions from N. coucang, an approximately 18 kDa protein composed of two 70-90 amino-acid chains linked by one or more disulfide bonds. The N-termini of these peptide chains exhibit nearly 70% sequence similarity (37% identity, chain 1; 54% identity, chain 2) with the two chains of Fel d 1, the major allergen from the domestic cat (Felis catus). The extensive sequence similarity between the brachial organ component of N. coucang and the cat allergen suggests that they exhibit immunogenic cross-reactivity. This work clarifies the chemical nature of the brachial organ exudate and suggests a possible mode of action underlying the noxious effects of slow loris bites.

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods.

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification.