5,6-Dichloro-1-methylgramine, a non-toxic antifoulant derived from a marine natural product.

The laboratory culture of the barnacle, Balanus amphitrite has made it possible to supply cypris larvae for antifouling assays all year round. The settlement of cyprids obtained from cultured B. amphitrite was indistinguishable from cyprids reared from field-collected barnacles. In laboratory cyprid settlement assays of extracts from marine sessile organisms, antifouling activity was expressed as the 99% inhibitory concentration (IC99), and toxicity as the 30% lethal concentration (LC30). The lipophilic extract of the marine bryozoan, Zoobotryon pellucidum, which showed promising antifouling activity, yielded 2,5,6-tribromo-1-methylgramine (TBG) by bioassay-guided isolation. The inhibitory activity of TBG was 6 times as strong as that of bis-(n-butyltin)oxide (TBTO), while its toxicity to cypris larvae was one-tenth that of TBTO. A structure-activity relationship study with 155 indole derivatives led to the discovery of the non-toxic antifoulant candidates 5,6-dichlorogramine, 5-chloro-2-methylgramine, and 5,6-dichrolo-1-methylgramine (DCMG), the latter being selected as the antifouling paint ingredient for performance evaluation tests (panel tests) following the results of a preliminary safety tests. A silicone-based antifouling paint containing 5-10% of DCMG was prepared and tested in the field; the painted surfaces remained almost barnacle-free for 1.5 years similar to silicone coatings such as Biox. Since the leaching rate of DCMG from the paint surface could be controlled by the addition of an acrylic acid-styrene copolymer (ASP), the life of the antifouling performance is expected to be improved. Thus, an extremely non-toxic silicone-based antifouling paint containing DCMG is under development.

Less damaging effect of whisky in rat stomachs in comparison with pure ethanol. Role of ellagic acid, the nonalcoholic component.

BACKGROUND/AIM: Ellagic acid (EA), one of the polyphenols that are abundantly contained in whisky as a nonalcoholic component, has antioxidant and anti-inflammatory activities. In the present study, we compared the action of whisky and pure ethanol on the rat gastric mucosa, and examined the role of EA in the less-damaging effect of whisky in the stomach. METHODS: Under urethane anesthesia, a rat stomach was mounted in an ex vivo chamber, perfused with saline, and the transmucosal potential difference (PD) was measured before and after exposure to whisky (Yamazaki, Suntory) and ethanol (43%). In a separate study, the animals were given whisky or ethanol (1 ml, 43%) p.o. under unanesthetized conditions, killed 1 h later, and the gastric mucosa was examined for hemorrhagic lesions. RESULTS: Both whisky and ethanol caused a PD reduction, resulting in damage in the stomach, but these responses were less marked in the case of whisky. Although the reduced PD recovered gradually after removal of ethanol, this process was significantly expedited by co-application of EA (80 microg/ml), the recovery rate being much the same as that observed after exposure to whisky. The less-damaging effect of whisky was confirmed in unanesthetized rats after p.o. administration of these agents. In addition, EA (1-30 mg/kg), administered p.o. together with absolute ethanol (99.9%), reduced the severity of gastric lesions induced by ethanol, in a dose-dependent manner, and the effect at 30 mg/kg was equivalent to that obtained by the whisky component containing several low- and high-molecular-weight polyphenols. EA had a scavenging action against both oxygen and hydroxyl radicals in vitro, the effect being equivalent to that of catechol or alpha-tocopherol. CONCLUSION: These results suggest that whisky is less irritating to the gastric mucosa, as compared with pure ethanol, and this property of whisky may be explained by EA, one of polyphenols contained in whisky, and its radical scavenging action.

Activation of torularhodin production by Rhodotorula glutinis using weak white light irradiation.

The effects of the irradiation of weak white light on the growth of the red yeast Rhodotorula glutinis and its production of carotenoids were investigated. The ability of beta-carotene and torularhodin, which are final products of carotenoid biosynthesis in R. glutinis, to quench singlet oxygen has also been investigated. Weak white light irradiation that has no effect on the growth of Saccharomyces cerevisiae inhibited the growth of R. glutinis. Simultaneously, the production of torularhodin by R. glutinis markedly increased. In a mutant of R. glutinis, which exhibited increased production of torularhodin, an increase in torularhodin production was shown as a result of light irradiation during the logarithmic growth phase. An experiment using 3-(1,4-epidioxyl-4-methyl-1,4-dehydro-1-naphtyl) propionic acid clarified that torularhodin inhibited 2,5-diphenyl-3,4-benzofran decomposition by singlet oxygen quenching more strongly than did beta-carotene. This result is consistent with the report that carotenoids having a longer polyene chain may exhibit a more potent ability to quench singlet oxygen. These results suggest that the biosynthesis of carotenoids in R. glutinis may play an important role in protecting against oxidative damage caused by light irradiation, and in particular, torularhodin which has a potent singlet oxygen quenching ability may be important. We suggest that acquisition of the ability to produce torularhodin may be an important property for this yeast to promote its wider distribution in the natural world.

Properties of a high-torularhodin-producing mutant of Rhodotorula glutinis cultivated under oxidative stress.

To characterize the properties of torularhodin, which is one of the carotenoid pigments produced by the yeast Rhodotorula sp., a mutant which produces large amounts of torularhodin was constructed and its tolerance against oxidative stress was investigated. The mutant we obtained was capable of producing large amounts of torularhodin in response to irradiation with blue light. The mutant, incubated under irradiation with white light that resulted in an increased production of torularhodin, exhibited resistance to growth inhibition induced by the addition of methylene blue as the generator of singlet oxygen. Leakage of lactate dehydrogenase to the growth medium from the mutant was not increased as compared to that from a parent strain and a high-beta-carotene-producing mutant. These results suggest that an increase in the production of torularhodin reduces the susceptibility to injury induced by an active oxygen species.

Inhibition of low-density lipoprotein oxidation by astaxanthin.

Marine animals produce astaxanthin which is a carotenoid and antioxidant. In this study we determined the in vitro and ex vivo effects of astaxanthin on LDL oxidation. The oxidation of LDL was measured in a 1 ml reaction system consisting of increasing concentrations of astaxanthin (12.5, 25.0, 50.0 microg/ml), 400 microM V-70 (2, 2'-azobis(4-methoxy-2, 4-dimethylvaleronitrile)), and LDL (70 microg/ml protein). Astaxanthin dose, dependently significantly prolonged the oxidation lag time (31.5, 45.4, 65.0 min) compared with the control (19.9 min). For the ex vivo study 24 volunteers (mean age 28.2 [SD 7.8] years) consumed astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg per day for 14 days. No other changes were made in the diet. Fasting venous blood samples were taken at days 0, +14. LDL lag time was longer (5.0, 26.2, 42.3 and 30.7% respectively) compared with day 0 after consuming astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg for 14 days compared with day 0, but there was no difference in oxidation of LDL between day 0 (lag time 59.9+/-7.2 min) and day 14 (57.2+/-6.0 min) in the control group. Our results provide evidence that consumption of marine animals producing astaxanthin inhibits LDL oxidation and possibly therefore contributes to the prevention of atherosclerosis.

Identification of the major antioxidative metabolites in biological fluids of the rat with ingested (+)-catechin and (-)-epicatechin.

(+)-Catechin and (-)-epicatechin are known to be biologically effective antioxidants present in the human diet, particularly in wine and tea. We studied the metabolism of these compounds to elucidate the truly active structures in biological fluids by their oral administration to rats. Without any treatment with beta-glucuronidase and sulfatase, a pair of metabolites were detected at much higher concentrations in the plasma, bile, and urine than the originally ingested compounds. Each major metabolite found in the plasma at the highest concentration was excreted in both the bile and urine, and was purified from urine. Their chemical structures were established to be (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide by MS and NMR analyses. These glucuronide conjugates exhibited high antioxidative activities as superoxide anion radical scavengers like their parent compounds. It is concluded that (+)-catechin 5-O-beta-glucuronide and (-)-epicatechin 5-O-beta-glucuronide are the biologically active in vivo structures of the ingested polyphenolic antioxidants.

Structure and function of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs.

Jelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish, Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8 and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.

New Trihydroxy-keto-carotenoids Isolated from an Astaxanthin-producing Marine Bacterium.

Polar orange pigments were extracted from the cultured cells of marine bacterium strain SD-212 and purified by chromatographic methods. The structures of two new trihydroxy-keto-carotenoids, (2R,3S,3'S)-2-hydroxyastaxanthin (1) and (2R,3S,3'R)-2-hydroxyadonixanthin (2) were determined by means of spectral methods. Known carotenoids (3S,2'R,3'R)-erythroxanthin (3), (2R,3S',2' R,3'S)-2,3,2',3'-tetrahydroxy-ß,ß-carotene-4,4'-dione (4), (2R,3S,2'R,3'R)-2,3,2',3'-tetrahydroxy-ß,ß-caroten-4-one (5), (3S,3'S)-astaxanthin (6), and (3S,3'R)-adonixanthin (7) were also isolated.

Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.

Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon beta-carotene by a single gene.

Compounds that include (a) keto group(s) in a molecule are ubiquitous natural components. A novel gene involved in ketocompound biosynthesis, designated crtW, was isolated from the marine bacteria Agrobacterium aurantiacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin. When this gene was introduced into Escherichia coli that accumulated beta-carotene due to the Erwinia carotenogenic genes, the E. coli transformants synthesized canthaxanthin, one of ketocarotenoids, which was identified after purification by its visible, FD-MS and 1H-NMR spectral analysis. It has been demonstrated for the first time that one gene encodes an enzyme "ketolase" that catalyzes the conversion of methylene groups of a hydrocarbon beta-carotene to keto groups for synthesizing canthaxanthin via echinenone.

Further identification of bioactive peptides in the anterior byssus retractor muscle of Mytilus: two contractile and three inhibitory peptides.

1. Two contractile and three inhibitory peptides were newly isolated from the anterior byssus retractor muscles (ABRMs) of the bivalve mollusc Mytilus edulis by using the muscle as the bioassay system. 2. The structures of the two contractile peptides were GPFGTHIKamide (GPFG-8) and GPFGLNKHGamide (GPFG-9). The contractile response of the ABRM to the first-time application of GPFG-8 or GPFG-9 was of considerable size. The threshold concentrations of the peptides were around 10(-9) M. However, the contractile response to the second-time application was far smaller than that to the first-time application in both cases. Namely, the muscle showed tachyphylaxis to the peptides. 3. Two of the three inhibitory peptides were members of the Mytilus-inhibitory-peptide (MIP) family. Their structures were RAPLFIamide (MIP6) and RSPMFVamide (MIP7). The peptides, as well as the other MIPs previously identified, showed a potent inhibitory effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. The remaining one was an MIP-related peptide (MIP-RP) having the sequence of MRYFVamide. The MIP-RP was less potent than the two MIPs in inhibiting the contraction of the ABRM.

Two S-Iamide Peptides, AKSGFVRIamide and VSSFVRIamide, Isolated from an Annelid, Perinereis vancaurica.

Two peptides, H-Ala-Lys-Ser-Gly-Phe-Val-Arg-Ile-NH2 (AKSGFVRIamide), and H-Val-Ser-Ser-Phe-Val-Arg-Ile-NH2 (VSSFVRIamide) were isolated from a polychaete annelid, Perinereis vancaurica. Both the peptides evoked rhythmic contractions in the esophagus of Perinereis with a threshold as low as 10-10-10-9 M, suggesting that the peptides may be involved in the regulation of gut motility of the animal. The sequences of these peptides are very similar to those of other S-Iamide family peptides which have been previously isolated from an echiuroid worm and some molluses. In particular, the sequence of VSSFVRIamide is identical to that of an echiuroid S-Iamide peptide. All of the molluscan and echiuroid S-Iamide peptides, as well as the annelid peptides, were found to produce contractions in the esophagus of Perinereis. On the other hand, the annelid S-Iamide peptides, as well as the molluscan and echiuroid peptides, were found to inhibit or potentiate contractions elicited by electrical stimulation in echiuroid and molluscan muscles. S-Iamide peptides may be a typical neuropeptide family distributed interphyletically in the Protostomia.

Physiological role of 3-hydroxykynurenine and xanthurenic acid upon crustacean molting.

Studies with crabs (Charybdis japonica) and crayfish (Procambarus clarkii) revealed that the tryptophan metabolites, 3-hydroxy-L-kynurenine (3-OH-K) and xanthurenic acid (XA), common secretory products of the X-organ-sinus gland complex of eyestalks from several decapods, regulated the molting of crustaceans in species-nonspecific fashion. Injection of 3-OH-K to the eyestalk-ablated crayfish delayed the onset of the first molt and lengthened the interval between the first and second molts. These lines of evidence were in accord with previous accounts of the so-called "molt inhibiting hormone" (MIH) effect. Removal of eyestalks caused a change in the conversion capacity of exogenous 3-OH-K to XA in the hemolymph. The peak in transformation capacity was followed by a peak in the titer of 20-hydroxyecdysone or molting hormone. Moreover, the seasonal profiles of the XA and ecdysone titers in Charybdis japonica exhibited a staggered relationship in the tissues tested. The ratio of XA to 3-OH-K, which is expected to indicate the apparent 3-OH-Kase activity, fluctuated seasonally and locally. When the Y-organ with the adhering tissues (Y-organ complex or YOC) was incubated during the period of high XA titer, the YOC produced 100 times more ecdysone than before incubation. It is suggested that ecdysteroidogenesis in situ was suppressed during this period by XA, but incubation of the YOC lead to a dramatic acceleration in ecdysone synthesis by overriding this inhibitory effect. XA profoundly repressed ecdysteroidogenesis in the YOC culture. Thus, XA is the ecdysone biosynthesis inhibitor (EBI) and 3-OH-K the precursor in crustaceans. An interfering effect of XA to a biocatalyst cytochrome P-450 system was postulated for the inhibition mechanism of ecdysteroidogenesis.

What is molt-inhibiting hormone? The role of an ecdysteroidogenesis inhibitor in the crustacean molting cycle.

The in vivo molt-inhibitory effects of the ecdysone biosynthesis inhibitors 3-hydroxy-L-kynurenine and xanthurenic acid were investigated. These ecdysone biosynthesis inhibitors, isolated from the eyestalks of blue crabs (Callinectes sapidus), were injected into eyestalk-ablated crayfish (Procambarus clarkii). The active factor was found to be species-nonspecific within crabs and crayfish. The seasonal profiles of the xanthurenic acid and ecdysone titers exhibited a staggered relationship. Moreover, the activity of a 3-hydroxy-L-kynurenine aminotransferase varied during the molting cycle. The data suggested that 3-hydroxy-L-kynurenine, which is secreted from the X-organ-sinus gland complex of crustaceans, is released into the hemolymph, and after accumulating at the surface of the Y-organ, is converted into the active form, xanthurenic acid. Xanthurenic acid was found to profoundly repress ecdysteroidogenesis in vitro.

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus.

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.

Endogenous inhibitor of ecdysone synthesis in crabs.

Attempts to isolate the molt-inhibiting hormone (MIH) of crustaceans from crab eyestalks (ES) resulted in the characterization of xanthurenic acid as an inhibitor of ecdysone biosynthesis in the cultured Y-organ-complex (YOC) homogenate. It was also found that 3-hydroxy-L-kynurenine present in the ES is transformed into xanthurenic acid in the YOC and body fluid. Its mode of inhibitory action in ecdysone biosynthesis is probably inactivation of cytochrome P-450.

Comparison of carotenoids in the ovaries of marine fish and shellfish.

1. The following carotenoids were isolated and identified: astaxanthin diester, tunaxanthin monoester, astaxanthin monoester, tunaxanthin, astaxanthin, doradexanthin, lutein, zeaxanthin, idoxanthin, triol and tetrol from nine species of fish; astaxanthin diester, astaxanthin monester, astaxanthin, doradexanthin, zeaxanthin, idoxanthin and tetrol from four species of crustacean, astaxanthin, pectenolone, pectenoxanthin, pectenol and tetrol from four species of scallop. 2. Tunaxanthin monoester and astaxanthin diester were major carotenoids in skipjack and Pacific cod, respectively. 3. The concentration of carotenoids ranged 0.065-1.95, 1.30-5.91 and 1.56-7.15 mg per 100 g ovary for fish, crustacean and scallop, respectively. 4. The species- and tissue-specificity of ovarian carotenoids and their possible role are discussed.