Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Apo-12'-capsorubinal exhibits anti-inflammatory effects and activates nuclear factor erythroid 2-related factor 2 in RAW264.7 macrophages.

Apocarotenoids have short carbon chain structures cleaved at a polyene-conjugated double bond. They can be biosynthesized in plants and microorganisms. Animals ingest carotenoids through food and then metabolize them into apocarotenoids. Although several apocarotenoids have been identified in the body, their precise health functions are still poorly understood. This study investigated the anti-inflammatory activities of apo-12'-capsorubinal in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. It was confirmed that apo-12'-capsorubinal was not cytotoxic to the macrophages at the concentrations tested. Apo-12'-capsorubinal treatment led to a marked downregulation of interleukin (IL)-6 protein and Il6 mRNA levels. This apocarotenoid exhibited more potent inhibitory effects than its parent carotenoids, capsanthin and capsorubin. Furthermore, apo-12'-capsorubinal, but not its parent carotenoids, promoted the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated the expression of Nrf2-target genes, such as heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1), in a dose-dependent manner. Furthermore, a comparison using apo-12'-zeaxanthinal and 7,8-dihydro-8-oxo-apo-12'-zeaxanthinal revealed that the α, ß-unsaturated carbonyl group on the polyene linear chain mediated the enhanced nuclear Nrf2 translocation, HO-1 expression, and inhibition of IL-6 production. In contrast, apo-12'-mytiloxanthinal, which harbored a hydroxyl group at C-8 of apo-12'-capsorubinal, did not exhibit any of these activities. These results indicated that the ß carbon of the α, ß-unsaturated carbonyl group in the linear part of the polyene chain is crucial to the Nrf2-activating and anti-inflammatory effects of apo-12'-capsorubinal. This study will advance our knowledge of the physiological significance of xanthophyll-derived apocarotenoids and their potential use as nutraceuticals and pharmaceuticals.

Excitation relaxation dynamics of carotenoids constituting the diadinoxanthin cycle.

Carotenoids (Cars) exhibit two functions in photosynthesis, light-harvesting and photoprotective functions, which are performed through the excited states of Cars. Therefore, increasing our knowledge on excitation relaxation dynamics of Cars is important for understanding of the functions of Cars. In light-harvesting complexes, there exist Cars functioning by converting the π-conjugation number in response to light conditions. It is well known that some microalgae have a mechanism controlling the conjugation number of Cars, called as the diadinoxanthin cycle; diadinoxanthin (10 conjugations) is accumulated under low light, whereas diatoxanthin (11 conjugations) appears under high light. However, the excitation relaxation dynamics of these two Cars have not been clarified. In the present study, we investigated excitation relaxation dynamics of diadinoxanthin and diatoxanthin in relation to their functions, by the ultrafast fluorescence spectroscopy. After an excitation to the S2 state, the intramolecular vibrational redistribution occurs, followed by the internal conversion to the S1 state. The S2 lifetimes were analyzed to be 175 fs, 155 fs, and 140 fs in diethyl ether, ethanol, and acetone, respectively, for diadinoxanthin, and 155 fs, 135 fs, and 125 fs in diethyl ether, ethanol, and acetone, respectively for diatoxanthin. By converting diadinoxanthin to diatoxanthin, the absorption spectra shift to longer wavelengths by 5-7 nm, and lifetimes of S2 and S1 states decrease by 11-13% and 52%, respectively. Differences in levels and lifetimes of excited states between diadinoxanthin and diatoxanthin are small; therefore, it is suggested that changes in the energy level of chlorophyll a are necessary to efficiently control the functions of the diadinoxanthin cycle.

Total Synthesis of Loroxanthin.

The first total synthesis of loroxanthin (1) was accomplished by Horner-Wadsworth-Emmons reaction of C25-apocarotenal 8 having a silyl-protected 19-hydroxy moiety with C15-phosphonate 25 bearing a silyl-protected 3-hydroxy-ε-end group. Preparation of apocarotenal 8 was achieved via Stille coupling reaction of alkenyl iodide 10 with alkenyl stananne 9, whereas phosphonate 25 was prepared through treatment of ally alcohol 23 with triethyl phosphite and ZnI2. The ally alcohol 23 was derived from the known (3R,6R)-3-hydroxy C15-aldehyde 20, which was obtained by direct optical resolution of racemate 20 using a semi-preparative chiral HPLC column.

Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon.

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Fucoxanthin inhibits hepatic oxidative stress, inflammation, and fibrosis in diet-induced nonalcoholic steatohepatitis model mice.

Nonalcoholic steatohepatitis (NASH) is associated with hepatocyte injury, excessive oxidative stress, and chronic inflammation in fatty liver, and can progress to more severe liver diseases, such as cirrhosis and hepatocellular carcinoma. However, currently there are no effective therapies for NASH. Marine carotenoid, fucoxanthin (Fx), abundant in brown seaweeds, has variable biological properties, such as anti-cancer, anti-inflammatory, anti-oxidative and anti-obesity. However, the effect of Fx on the development of NASH has not been explored. We investigated the protective effects of Fx in diet-induced NASH model mice fed choline-deficient L-amino acid-defined high fat diet (CDAHFD). Fx administration significantly attenuated liver weight gain and hepatic fat accumulation, resulting in the alleviation of hepatic injury. Furthermore, the Fx-fed mice, not only exhibited reduced hepatic lipid oxidation, but also decreased mRNA expression levels of inflammation and infiltration-related genes compared to that of the CDAHFD-fed mice. Moreover, fucoxanthinol and amarouciaxanthin A, two Fx metabolites exerted anti-inflammatory effects in the liver via inhibiting the chemokine production in hepatocytes. In case of fibrosis, one of the features of advanced NASH, the expression of fibrogenic factors including activated-hepatic stellate cell marker was significantly decreased in the liver of Fx-fed mice. Thus, the present study elucidated that dietary Fx not only inhibited hepatic oxidative stress and inflammation but also prevented early phase of fibrosis in the diet-induced NASH model mice.

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Carotenoid Profiling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales.

Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and ß-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (ß,ε-caroten-3'-ol), and zeinoxanthin (ß,ε-caroten-3-ol) were identified. In addition, ß-cryptoxanthin, an intermediate in zeaxanthin synthesis from ß-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and ¹H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Versatile Amine-Promoted Mild Methanolysis of 3,5-Dinitrobenzoates and Its Application to the Synthesis of Colorado Potato Beetle Pheromone.

A mild deacylation method for 3,5-dinitrobenzoates using methanolic solutions of amines, such as dialkylamines, was developed. The method's versatility was confirmed by applying it to synthesizing a key intermediate for Colorado potato beetle pheromone.

Anti-Oxidative Activity of Mytiloxanthin, a Metabolite of Fucoxanthin in Shellfish and Tunicates.

Anti-oxidative activities of mytiloxanthin, a metabolite of fucoxanthin in shellfish and tunicates, were investigated. Mytiloxanthin showed almost the same activities for quenching singlet oxygen and the inhibition of lipid peroxidation as those of astaxanthin, which is a well-known singlet oxygen quencher. Furthermore, mytiloxanthin showed excellent scavenging activity for hydroxyl radicals and this activity was markedly higher than that of astaxanthin.

Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

Synthesis of (3S,3'S)- and meso-stereoisomers of alloxanthin and determination of absolute configuration of alloxanthin isolated from aquatic animals.

In order to determine the absolute configuration of naturally occurring alloxanthin, a HPLC analytical method for three stereoisomers 1a-c was established by using a chiral column. Two authentic samples, (3S,3'S)- and meso-stereoisomers 1b and 1c, were chemically synthesized according to the method previously developed for (3R,3'R)-alloxanthin (1a). Application of this method to various alloxanthin specimens of aquatic animals demonstrated that those isolated from shellfishes, tunicates, and crucian carp are identical with (3R,3'R)-stereoisomer 1a, and unexpectedly those from lake shrimp, catfish, biwa goby, and biwa trout are mixtures of three stereoisomers of 1a-c.

Total synthesis of gobiusxanthin stereoisomers and their application to determination of absolute configurations of natural products: revision of reported absolute configuration of epigobiusxanthin.

(3R)-Gobiusxanthin stereoisomers (1a-d) were synthesized by stereoselective Wittig reaction of the (3R)-C15-acetylenic tri-n-butylphosphonium salt 7 with C25-apocarotenal stereoisomers 5a,b and 14a,b bearing four kinds of 3,6-dihydroxy-ε-end groups. The validity of the reported stereochemistry of gobiusxanthin was demonstrated by the fact that the reported spectral data of natural gobiusxanthin were in agreement with those of synthetic (3R,3'S,6'R)-gobiusxanthin (1a). On the other hand, the reported CD spectral data of natural epigobiusxanthin, which has been assigned as (3R,3'R,6'R)-isomer (3'-epigobiusxanthin), were identical with those of synthetic (3R,3'S,6'S)-isomer 1d (6'-epigobiusxanthin) rather than those of the corresponding synthetic 3'-epi-isomer 1b. It was found that the stereochemistry at C3-position has little effect on the shape of their CD spectra. Thus, in order to reinforce the validity of the absolute configurations at C3-position of natural specimens, (3S,3'S,6'R)- and (3S,3'S,6'S)-stereoisomers 1e and 1f were also synthesized and a HPLC analytical method for four stereoisomers was established by using a column carrying a chiral stationary phase. The HPLC analysis has proven that the stereochemistry of the natural epigobiusxanthin is 3R,3'S,6'S.

Identification and tissue distribution of fucoxanthinol and amarouciaxanthin A fatty acid esters in fucoxanthin-fed mice.

Administered carotenoid fatty acid esters are thought to be hydrolyzed to their free forms and absorbed into the body, and information on the tissue distribution of carotenoid fatty acid esters has been limited. Fucoxanthin, a marine carotenoid, exhibits various health benefits, including anti-diabetic and anti-obesity effects. However, fucoxanthin metabolism in mammals remains unclear. Herein, we investigated the fatty acid esters of fucoxanthin metabolites, fucoxanthinol and amarouciaxanthin A, in the tissues of male C57BL/6J mice fed a fucoxanthin-containing diet for one week. Fucoxanthinol and amarouciaxanthin A-3-esters accumulated abundantly in the liver and epididymal white adipose tissue, respectively. These esters were less detectable in the serum and other tissues. Therefore, it is suggested that fucoxanthinol and amarouciaxanthin A are partially acylated in the liver and epididymal white adipose tissue after being transported through the body as their free forms. This study presents a novel carotenoid metabolic pathway in mammals.

Chromophore Structure in an Inactive State of a Novel Photosensor Protein Opn5L1: Resonance Raman Evidence for the Formation of a Deprotonated Adduct at the 11th Carbon Atom.

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Targeting transglutaminase 2 mediated exostosin glycosyltransferase 1 signaling in liver cancer stem cells with acyclic retinoid.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

Stereoselective total synthesis of the acetylenic carotenoids alloxanthin and triophaxanthin.

Stereoselective total synthesis of the C(40)-diacetylenic carotenoid alloxanthin (1) and the C(31)-acetylenic apocarotenoid triophaxanthin (2) was accomplished by Wittig condensation of C(10)-dialdehyde 20 or C(16)-keto aldehyde 19, respectively, with C(15)-acetylenic tri-n-butylphosphonium salt 12.

Lipophilic amines as potent inhibitors of N-acylethanolamine-hydrolyzing acid amidase.

N-Acylethanolamines (NAEs) including N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine are endogenous lipid mediators. These molecules are degraded to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH) or NAE-hydrolyzing acid amidase (NAAA). Lipophilic amines, especially pentadecylamine (2c) and tridecyl 2-aminoacetate (11b), were found to exhibit potent NAAA inhibitory activities (IC(50)=5.7 and 11.8µM), with much weaker effects on FAAH. These simple structures would provide a scaffold for further improvement in NAAA inhibitory activity.