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A red alga, Chondria armata, was used as a vermifuge in Japan. Previously, we isolated domoic acids from C. armata guided by the insecticidal activity against American cockroaches. Further investigations led to the discovery of a compound one-thousand times more toxic than domoic acid. It showed lethal activity at 5 ng against cockroaches. The insecticidal compound was identified as palytoxin by means of NMR and MS. Careful interpretation of the NMR spectra allowed us to refine the previously reported NMR assignment of palytoxin. Palytoxin has been isolated from various organisms, and it is significant that palytoxin was isolated from marine plants.

The commercial quality of Japanese Angelica radices — Angelica acutiloba Kitagawa (Yamato-toki) and A. acutiloba Kitagawa var. sugiyama Hikino (Hokkai-toki) — used in Kampo traditional herbal medicines, was studied by use of omics technologies. Complementary and alternative medical providers have observed in their clinical experience that differences in radix commercial quality reflect the differences in pharmacological responses; however, there has been little scientific examination of this phenomenon. The approach of omics, including metabolomics, transcriptomics, genomics, and informatics revealed a distinction between the radix-quality grades based on their metabolites, gene expression in human subjects, and plant genome sequences. Systems biology, constructing a network of omics data used to analyze this complex system, is expected to be a powerful tool for enhancing the study of radix quality and furthering a comprehensive understanding of all medicinal plants.

We examined the bioactivity of Yamato-mana (Brassica rapa L. Oleifera Group) constituent glucosinolates and found that 3-butenyl glucosinolate (gluconapin) decreased the plasma triglyceride gain induced by corn oil administration to mice. However, phenethyl glucosinolate (gluconasturtiin) had little effect. 2-Propenyl glucosinolate (sinigrin) also reduced the plasma triglyceride level, which suggests that alkenyl glucosinolates might be promising agents to prevent postprandial hypertriglyceridemia.

Glycosylation is one of the key modification steps for plants to produce a broad spectrum of flavonoids with various structures and colors. A survey of flavonoids in the blue flowers of Veronica persica Poiret (Lamiales, Scrophulariaceae), which is native of Eurasia and now widespread worldwide, led to the identification of highly glycosylated flavonoids, namely delphinidin 3-O-(2-O-(6-O-p-coumaroyl-glucosyl)-6-O-p-coumaroyl-glucoside)-5-O-glucoside (1) and apigenin 7-O-(2-O-glucuronosyl)-glucuronide (2), as two of its main flavonoids. Interestingly, the latter flavone glucuronide (2) caused a bathochromic shift on the anthocyanin (1) toward a blue hue in a dose-dependent manner, showing an intermolecular co-pigment effect. In order to understand the molecular basis for the biosynthesis of this glucuronide, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT88D8), based on the structural similarity to flavonoid 7-O-glucuronosyltransferases (F7GAT) from Lamiales plants. Enzyme assays showed that the recombinant UGT88D8 protein catalyzes the 7-O-glucuronosylation of apigenin and its related flavonoids with preference to UDP-glucuronic acid as a sugar donor. Furthermore, we identified and functionally characterized a cDNA encoding another UGT, UGT94F1, as the anthocyanin 3-O-glucoside-2''-O-glucosyltransferase (A3Glc2''GlcT), according to the structural similarity to sugar-sugar glycosyltransferases classified to the cluster IV of flavonoid UGTs. Preferential expression of UGT88D8 and UGT94F1 genes in the petals supports the idea that these UGTs play an important role in the biosynthesis of key flavonoids responsible for the development of the blue color of V. persica flowers.

The monoterpene glycoside, 3'-O-galloylpaeoniflorin (1), and four known compounds, 6'-O-galloylalbiflorin (2), pentagalloylglucose (3), 6'-O-benzoylpaeoniflorin (4) and 6'-O-galloylpaeoniflorin (5), were isolated from the roots of Paeonia lactiflora that had been grown and processed in Nara prefecture, Japan, as androgen modulators. Their structures were elucidated based on spectroscopic analysis. Compounds 2 and 3 showed strong androgen receptor (AR) binding activity (IC(50) values 33.7 and 4.1 microg/ml, respectively), 1, 4 and 5 showed weak activity (20, 31 and 12% at 120 microg/ml, respectively). However, paeoniflorin (6) and albiflorin (7), the structures of which are related to 1, 2, 4 and 5, showed no activity. These results suggested that both the structure of albiflorin and the galloyl moiety are important for 2 to show strong AR binding activity. Furthermore, compounds 1-5 inhibited growth of an androgen-dependent LNCaP-FGC (prostate cancer cell line), and were indicated to be AR antagonists. Compounds 2 and 3 might be candidates as safe, natural anti-androgens.

Iron (Fe) is an essential element for plant growth. Gramineous plants have generally developed a distinct strategy to efficiently acquire insoluble Fe, which is characterized by the synthesis and secretion of an Fe-chelating substance, phytosiderophore (PS) such as mugineic acid (MA), and by a specific uptake system for Fe(III)-PS complexes. In a previous study, we identified a gene specifically encoding an Fe(III)-PS transporter (HvYS1) in barley. This gene as well as the encoded protein is specifically expressed in the epidermal cells of the roots, and gene expression is greatly enhanced under Fe-deficient conditions. The localization and substrate specificity of HvYS1 indicate that it is a specific transporter in barley roots. In contrast, ZmYS1, which has been reported as an Fe-PS transporter from maize, possesses broad substrate specificity despite a high homology with HvYS1. By assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, we revealed that the outer membrane loop between the 6th and 7th transmembrane regions is essential for the substrate specificity. We also achieved an efficient short-step synthesis of MA and 2'-deoxymugineic acid (DMA). Our new synthetic method enabled us to use them in a large quantity for biological studies.

Two novel blue pigments, rosacyanins A1 and A2, were isolated from the petals of Rosa hybrida cv. ‘M'me. Violet’. Their structures were elucidated on the basis of high-resolution Fourier transform ion cyclotron resonance mass spectroscopy (HR-FT-ICR-MS), FABMS/MS/MS, 1 H, 13 C and two-dimensional NMR. The molecular formulas of rosacyanin A1 ( 1 ) and A2 ( 2 ) are C 56 H 37 O 31 and C 63 H 41 O 35 , respectively. The structures of rosacyanins A1 and A2 consisted of a common chromophore containing cyanidin with a galloyl group link between positions 4 and 5 of the hydroxyl group of the flavylium nucleus and tellimagrandins (1 or 2). These pigments in which anthocyanidin nuclei linked to ellagitannin through an ether bond are the first compounds isolated from natural sources.

Tachykinins (TKs) constitute the largest vertebrate neuropeptide family with multifunctions in central and peripheral tissues. In several invertebrate species, two types of structurally related peptides, 'tachykinin-related peptides (TKRPs)' and 'invertebrate tachykinins (inv-TKs)' have been identified. TKRPs, isolated from the nerve and/or gut tissues, contain the common C-terminal sequence -Phe-X-Gly-Y-Arg-NH(2) (X and Y are variable) analogous to the vertebrate TK consensus -Phe-X-Gly-Leu-Met-NH(2), and exhibit vertebrate TK-like contractile activity on invertebrate gut tissues. Inv-TKs have been shown to be present exclusively in the salivary gland of several species, to share vertebrate TK consensus motif, and to possess TK-like potencies on vertebrate, not invertebrate tissues. However, the functional and evolutionary relevance of TKRPs and inv-TKs to vertebrate TKs remains to be understood. Recent studies have revealed that TKRP precursors dramatically differ from vertebrate preprotachykinins in structural organization and that TKRP receptors share structural and functional properties with vertebrate TK receptors. Moreover, the C-terminal arginine in TKRPs has been shown to play an essential role in discriminating their receptors from vertebrate TK receptors. Such recent marked progress is expected to enhance further investigation of biological roles of TKRPs. This review provides an overview of the basic findings obtained previously and a buildup of new knowledge regarding TKRPs and inv-TKs. We also compare TKRPs and inv-TKs to vertebrate TKs with regard to evolutionary relationships in structure and function among these structurally related peptides.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.

Urechistachykinin I and II (Uru-TK I and II) are invertebrate tachykinin-related peptides (TRPs), which have been isolated from echiuroid worms. The cDNA sequence encoding the Uru-TK I and II revealed that the precursor also encoded five TRP-like peptides. Here, we report the characterization of these Uru-TK-like peptides named as Uru-TK III-VII. Northern and Southern blot analyses demonstrated that Uru-TK mRNA is localized in nerve tissue. In addition, the presence of the Uru-TK-like peptides as matured forms in the nerve tissue was detected by mass spectrometric analysis, and identified these peptides were shown to exhibit a contractile activity on cockroach hindgut that was as potent as that of Uru-TK II. Furthermore, synthetic Uru-TK-like peptide analogs which contained Met-NH2 instead of Arg-NH2 at their C-termini were shown to possess a potential to bind to a mammalian tachykinin receptor, indicating that Uru-TK-like peptides are likely to correspond to vertebrate tachykinins, except for the difference at the C-terminal residue. These findings show that Uru-TK-like peptides are essentially equivalent to Uru-TK I and II, leading to the proposal that Uru-TK-like peptides play an essential role as invertebrate tachykinin neuropeptides.

Two new galloylated monoterpene glycosides, 4-O-galloylalbiflorin and 4'-O-galloylpaeoniflorin, were isolated from the roots of Paeonia lactiflora that had been grown and processed in Nara prefecture, Japan. Their structures were elucidated based on spectroscopic analysis. These compounds showed androgen receptor (AR) binding activity.

Phytosiderophores, mugineic acids, have been demonstrated to be involved in Fe acquisition in gramineous plants. In this study, chromosomal arm locations of genes encoding for biosynthesis of various phytosiderophores were identified in a cultivar of barley (Hordeum vulgare L. cv. Betzes). Using wheat (Triticum aestivum L. cv. Chinese Spring)-barley (cv. Betzes) ditelosomic addition lines for 4HS and 4HL, a gene for hydroxylation of 2′-deoxymugineic acid to mugineic acid was localized to the long arm of barley chromosome 4H. To locate the gene for hydroxylation of mugineic acid to 3-epihydroxymugineic acid, hybrids between the 4H addition line and other wheat-barley addition lines were studied. Only a hybrid between 4H and 7H addition lines produced 3-epihydroxymugineic acid. The gene was further localized to the long arm of chromosome 7H by feeding mugineic acid to ditelosomic addition lines for 7HS and 7HL. A new phytosiderophore was discovered in both 7H and 7HL addition lines, which was identified to be 3-epihydroxy-2′-deoxymugineic acid by detailed nuclear magnetic resonance studies. These results revealed that in barley there are two pathways from 2′-deoxymugineic acid to 3-epihydroxymugineic acid: 2′-deoxymugineic acid → mugineic acid → 3-epihydroxymugineic acid and 2′-deoxymugineic acid → 3-epihydroxy-2′-deoxymugineic acid → 3-epihydroxymugineic acid. Barley genes encoding for the hydroxylations of phytosiderophores are located in different chromosomes and each gene hydroxylates different C-positions: the long arm of chromosome 4H carries the gene for hydroxylating the C-2′ position and the long arm of chromosome 7H carries the gene for hydroxylating the C-3 position of the azetidine ring.

Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.

Two novel diacylated anthocyanins and two known monoacylated anthocyanins were isolated from violet petals of Petunia hybrida cv. Surfinia Violet Mini. Their structures were elucidated by spectral methods. The major novel pigment was identified as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)- p -coumaroyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β -d-glucopyranoside and the minor novel pigment was determined as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)-caffeoyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β - d-glucopyranoside.

Penostatins F–I have been isolated from a strain of Penicillium sp. originally separated from the marine alga Enteromorpha intestinalis, and their stereostructures have been established on the basis of spectral analyses. All the compounds exhibit significant cytotoxicity against cultured P388 cells.

The GGNG peptides are myoactive peptides which we have isolated from several species of annelids. Two types of peptides have been isolated as GGNG peptides: earthworm excitatory peptides (EEP) and leech excitatory peptide (LEP). The EEP and LEP are highly homologous with each other but act specifically on earthworms and leeches, respectively. To approach the mechanism of the specific action, we attempted to characterize the receptors for EEP in the earthworm Eisenia foetida, from which EEP have been isolated, by a radioreceptor assay. To prepare a radiolabeled ligand, we first coupled EEP to Bolton-Hunter reagent and then labeled it with 125I ([125I] BH-EEP). Binding of the [125I] BH-EEP to the membrane preparation of the earthworm tissue (crop-gizzard) was reversible, saturable, and specific with a Kd of 4.9 ± 1.2 nM and a Bmax of 15.9 ± 2.0 fmol/mg wet tissue. For the earthworm membrane preparation, EEP showed a much greater potency than LEP in displacing [125I] BH-EEP, suggesting that the earthworm tissues contain EEP-specific receptors. This result would account for the specific biological activity of EEP and LEP on the earthworm and leech tissues, respectively. The binding capacity was high in the anterior part of digestive tract including the esophagus, crop and gizzard, and the nephridia. From these results, together with the observed myotropic activity of EEP on gut tissues, it is assumed that EEP is physiologically involved in regulation of the gut motility in the earthworm E. foetida.J. Exp. Zool. 279:562–570, 1997.© 1997 Wiley-Liss, Inc.

The quantity and localization of Eisenia tetradecapeptide which was isolated from the gut of the earthworm Eisenia foetida were examined in tissues of the same species by enzyme-linked immunosorbent assay and immunohistochemistry. Analysis by enzyme-linked immunosorbent assay showed that Eisenia-tetradecapeptide-like immunoreactivity was present in both the central nervous system (cerebral ganglion, subesophageal ganglion, ventral ganglia, and ventral nerve cord) and the gut (esophagus, crop, gizzard, and intestine). The central nervous system contained a higher amount of Eisenia-tetradecapeptide-like immunoreactivity (1.3 pmol/mg wet weight) than the gut (0.2-0.6 pmol/mg wet weight). Eisenia-tetradecapeptide-like immunoreactivity was scarcely detected in the body-wall muscle, nephridia, and sexual organs (testis, ovary, seminal vesicle, and ovisac). Immunohistochemical analysis demonstrated that intense Eisenia-tetradecapeptide-like immunopositive cells and nerve fibers were present in the central nervous system. Immunoreactivity was found in the epithelial cells lining the esophagus and in the submucous plexus in various parts of the gut. Thus, the present study suggests that Eisenia tetradecapeptide is a neuropeptide and/or peptide hormone present in both the central nervous system and the gut of the earthworm and that its role involves the regulation of gut motility.

An internal detoxification mechanism for Al was investigated in an Al-accumulating plant, hydrangea (Hydrangea macrophylla), focusing on Al forms present in the cells. The leaves of hydrangea contained as much as 15.7 mmol Al kg-1 fresh weight, and more than two-thirds of the Al was found in the cell sap. Using 27Al- nuclear magnetic resonance, the dominant peak of Al was observed at a chemical shift of 11 to 12 parts per million in both intact leaves and the extracted cell sap, which is in good accordance with the chemical shift for the 1:1 Al-citrate complex. Purification of cell sap by molecular sieve chromatography (Sephadex G-10) combined with ion-exclusion chromatography indicated that Al in fractions with the same retention time as citric acid contributed to the observed 27Al peak in the intact leaves. The molar ratio of Al to citric acid in the crude and purified cell sap approximated 1. The structure of the ligand chelated with Al was identified to be citric acid. Bioassay experiments showed that the purified Al complex from the cell sap did not inhibit root elongation of corn (Zea mays L.) and the viability of cells on the root tip surface was also not affected. These observations indicate that Al is bound to citric acid in the cells of hydrangea leaves.

We recently isolated an oviposition-inducing peptide that was identified as avian galanin from the oviducts of the Japanese quail. Avian galanin was localized in neural fibers distributed in muscle layers in the uterine and vaginal oviduct regions, and potentiated spontaneous contractions of the uterus and vagina. To elucidate whether an oviposition-inducing effect of avian galanin is due to the direct action on the oviduct, therefore, a specific binding site for avian galanin was determined in the functional quail oviduct in this study. The binding of [125I]iodoavian galanin was primarily located in the oviduct as well as the brain. The galanin binding was specifically inhibited as a function of the concentrations of both avian and rat galanins. The specific binding of avian galanin to the quail oviduct was temperature dependent and reached the maximum level for 1 h at 20 degrees C. In several regions of the oviduct, a higher level of specific galanin binding was observed only in the uterus and vagina. In contrast, the specific binding was low in the isthmus and negligible in the magnum. A similar localization was evident in the functional chicken oviduct. The Scatchard plot analysis of the binding of avian galanin to the uterine preparation revealed that the dissociation constant (Kd) was 0.249 (95% confidence interval, 0.192-0.356) nM, and the number of binding sites was 1.13 (0.99-1.36) fmol per mg tissue, respectively. During development, the galanin-binding sites were apparent in the quail oviduct at 3 weeks of age and the number of binding sites markedly increased between 3 weeks and 3 months of age. However, there was no significant change in the Kd value in the developing quail oviduct. This is the first demonstration of the presence of galanin receptors in the reproductive tract, such as the uterine and vaginal oviduct. The present results suggest that the number of galanin receptors in the oviduct increases during development and that galanin acts directly on the mature uterus and vagina to induce their contractions. This mechanism may be essential to the avian oviposition.

Annetocin, an oxytocin-related peptide which we isolated from the earthworm Eisenia foetida, induced a series of egg-laying-related behaviors in the earthworms. These stereotyped behaviors consisted of well-defined rotatory movements, characteristic body-shape changes, and mucous secretion from the clitellum. Each of these behaviors is known to be associated with formation of the cocoon in which eggs are deposited. In fact, some of the earthworms injected with annetocin (> 5 nmol) laid eggs. Such egg-laying-related behaviors except for oviposition were also induced by oxytocin, but not by Arg-vasopressin and some other bioactive peptides isolated from E. foetida. Furthermore, annetocin also induced these egg-laying-like behaviors in the leech Whitmania pigra, but not in the polychaete Perinereis vancaurica. These results suggest that annetocin plays some key role in triggering stereotyped egg-laying behaviors in terrestrial or fresh-water annelids that have the clitella.

Discovery of mugineic acids as phytosiderophores has shown that some graminaceous monocotyledonous plants have a different iron acquisition strategy (strategy II) from dicotyledonous and nongraminaceous monocotyledonous plants (strategy I). The process of iron acquisition by strategy II plants can be divided into four main steps: biosynthesis, secretion, solubilization, and uptake, all of which are effectively regulated by different systems. The biosynthesis of mugineic acids is controlled by an on-off system which is operated under the control of iron demand in the plant. All mugineic acids share the same biosynthetic pathway from L-methionine to 2'-deoxymugineic acid, but the subsequent steps differ among plant species and even cultivars. The biosynthesis of mugineic acids is associated with the methionine recycling pathway. The secretion of mugineic acids shows a distinct diumal rhythm. Mugineic acids solubilize sparingly soluble inorganic iron by chelation and possess a high chelation affinity for iron, but not for other polyvalent ions such as Ca 2+ , Mg 2+ and Al 3+ . The iron uptake process is regulated by a specific uptake system that transports the mugineic acid-Fe(III) complex as an intact molecule. This system specifically recognizes the mugineic acid-Fe(III) complexes, but not other mugineic acid-metal or synthetic chelator-Fe(III) complexes, suggesting that binding sites with strict recognition for stereostructure of the complex are located on the plasma membrane. All these regulatory systems are considered to represent an efficient strategy to acquire adequate amounts of iron and to avoid factors unfavorable for iron acquisition such as high pH, high concentrations of bicarbonate, Ca 2+ and Mg 2+ , microbial degradation, and uptake of other metals that are common in calcareous soils.

The isolated anterior gut (crop-gizzard) of the oligochaete annelid, Eisenia foetida, was used as a bioassay system to identify the bioactive peptides that regulate rhythmic spontaneous contractions of the gut. Three pentapeptides were purified from the extract of the gut (crop, gizzard and intestine) of the earthworm. These peptides showed inhibitory actions on gut motility and were termed Eisenia inhibitory pentapeptides (EIPPs). EIPPs had the most remarkable effect on the crop among the various parts of the gut with a threshold concentration as low as 10−10–10−9 M. From the pharmacological results, the inhibitory actions of EIPPs on the gut motility seem to be different from those of FMRFamide. The amino acid sequences of EIPPs were very similar to certain molluscan and echiuroid peptides previously identified. This is the first isolation of annelid peptides inhibiting gut motility except for FMRFamide.

Penostatins A ( 1 ), B ( 2 ), C ( 3 ) and ( D ) have been isolated from a strain of Penicillium sp. originally separated from the marine alga Enteromorpha intestinalis , and their stereostructures have been established on the basis of spectral analyses. The compounds 1∼3 exhibited significant cytotoxicity against cultured P388 cells.

The chemical structure of imidazolopyrroloquinoline (IPQ), a main reaction product of pyrroloquinolinequinone and L-tryptophan in vitro, has been determined by X-ray crystal analysis. The IPQ molecules exist in keto form, having three neutral carboxyl groups, and they form continuous and prominent stacking structures in a crystal which are stabilized by van der Waals, dipole-dipole and electrostatic interactions. There are three crystallographically independent dimethyl sulfoxide solvents in a crystal, and they stabilize the stacking layers of IPQ molecules by intermolecular hydrogen bonds between neighboring oxygen atoms.

Three novel bioactive peptides, which were structurally related to each other, were purified and chemically identified from two species of the earthworms, Eisenia foetida and Pheretima vittata. One peptide was isolated from the gut tissue of E. foetida, and the other two were purified from the whole body of E. foetida and P. vittata, respectively. These peptides consisted of 17 or 18 amino acid residues and were named GGNG peptides after their unique, common structure of the C-termini. These GGNG peptides augmented both tension and frequency of spontaneous contractions of isolated gut preparations of E. foetida, and also elicited contractions of other annelidan tissues such as the polychaete esophagus and the leech vagina. However, they showed no activity on molluscan or arthropodan tissues, suggesting that GGNG peptides may be peculiar to annelids. No peptides homologous to GGNG peptides have been known so far in any living organisms.

To elucidate the biosynthetic pathways of 3-epihydroxymugineic acid and 3-hydroxymugineic acid, feeding experiments using 2H- and/or 13C-labeled compounds were conducted with barley (Hordeum vulgare L. cv Ehimehadaka) and rye (Secale cereale L. cv Elubon), respectively. Feeding of [1,4′,4″-13C3]2′-deoxymugineic acid (25 atom% 13C) to iron-deficient barley yielded mugineic acid and 3-epihydroxymugineic acid enriched with 13C at their corresponding positions. These results enabled us to demonstrate that 2′-deoxymugineic acid acts as a precursor for mugineic acid and 3-epihydroxymugineic acid. The 2H-labeled 3-hydroxymugineic acid was obtained by feeding d,l- [3,3,4,4-2H4] methionine (98.6 atom% 2H) to iron-deficient rye plants. The 2H-NMR study revealed that two deuterium atoms (one each at the C-2′ and C-3 positions) from the three 2H-labeled methionine molecules were lost in 3-hydroxymugineic acid, whereas the other 10 deuterium atoms were incorporated in a manner similar to that of 2′-deoxymugin...

An oxytocin-vasopressin-related peptide, Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2, was isolated from the lumbricid earthworm, Eisenia foetida and termed annectocin. Annetocin potentiated not only spontaneous contractions of the gut but also pulsatory contractions and bladder-shaking movement of the nephridia. Annetocin may be involved in osmoregulation of the animal through nephridial function.

Achatin-I (Gly-D-Phe-Ala-Asp) is a tetrapeptide isolated from the ganglia of an African giant snail (Achatina fulica Férussac). The compound and its derivatives were synthesized, and their effects on the action potential of the crayfish giant axon elicited by electrical stimulation were examined by an intracellular recording method. The membrane potential after the action potential had fallen was elevated by some compounds at an acidic but not neutral pH level. An analysis of the structure-activity relationship for the activity to elevate the afterpotential at an acidic pH level, suggests that the factors favorable for activity were the beta-turn conformation, a beta- or gamma-carboxyl group in the C-terminal residue, appropriate hydrophobicity and/or bulkiness in the protecting groups for the alpha-amino group at the N-terminus, and the number of negative charges in the entire molecule.

A novel violet pigment, rosacyanin B, was isolated from the petals of Rosa hyb rida cv. ‘M'me Violet’, and its structure was elucidated by spectroscopic methods. rosacyanin B is the first C-4 substituted anthocyanidin isolated from intact plants.

This study provides new information on the Fe uptake system capable of supporting growth of the organism. Pseudomonas fluorescens isolated from the rhizosphere of barley, a gramineous plant, produced a siderophore under iron-limiting conditions. Its chemical structure was identified as pyochelin, on the basis of 1H and 13C NMR data of a stable methyl ester derivative. The same iron-limiting conditions induced a new set of outer membrane proteins (75 and 55 kDa), consistent with a siderophore-mediated iron-uptake system.

The interfering effects of copper, zinc, and cobalt on the uptake of mugineic acid-ferric complex were studied in barley (Hordeum vulgare, cv. Minorimugi) grown in nutrient solution. Short-term uptake experiments of 3 h were performed utilizing both ionic and mugineic acid-complex forms of each metal at two different concentrations. Copper was most effective in decreasing iron uptake when added in an ionic form at either concentration. The inhibition order at higher concentrations followed Cu(II) > Zn(II) ≥ Co(II), Co(III), which is consistent with the stability constants of these metal complexes with mugineic acid. The displacement of iron from its mugineic acid complex by these metals is suggested as a probable explanation for the decreased iron uptake. The inhibitory effect of metal complexes with mugineic acid on iron uptake was only found in cases with higher concentrations of Cu(II) and Zn(II) complexes. Deformation of the specific iron transport system in the plasma membrane due to their adsorption may be responsible for this effect.

The uptake rate by iron-deficient barley roots ( Hordeum vulgare , cv Minorimugi) from mugineic acid complexed with Fe(III) was much higher than corr

Two novel neuropeptides, urechistachykinin I (H-Leu-Arg-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH2) and urechistachykinin II (H-Ala-Ala-Gly-Met-Gly-Phe-Phe-Gly-Ala-Arg-NH2), were isolated from the ventral nerve cords of the echiuroid worm, Urechis unicinctus. These peptides showed a contractile action on the inner circular body-wall muscle of the animal. Their amino acid sequences were found to be significantly homologous with those of the vertebrate and insect tachykinins. The urechistachykinins potentiated spontaneous rhythmic contractions of the cockroach hindgut.

1. An SCP-related peptide, Ala-Pro-Asn-Phe-Leu-Ala-Tyr-Pro-Arg-Leu-NH2, was isolated from the ABRMs of Mytilus edulis. The peptide was designated Mytilus SCP. 2. At 10(-10) M or higher, Mytilus SCP showed a potentiating effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. In contrast, the peptide did not show any potentiating effect on contractures in response to ACh and the FMRFamide-related Mytilus decapeptide, suggesting that the potentiating effect on phasic contraction was brought about by an action of the peptide on the nerve elements in the ABRM. 3. At 10(-8) M or higher, Mytilus SCP showed a catch-relaxing effect in addition to the potentiating effect. The relaxing effect was blocked by mersalyl, suggesting that it was also brought about by a presynaptic action. 4. Various analogues of Mytilus SCP were examined on the ABRM. Leu-Ala-Tyr-Pro-Arg-Leu-NH2 was suggested to be the minimum structure required for both potentiating and relaxing activities on the ABRM. Leu-D-Ala-Tyr-Pro-Arg-D-Leu-NH2 and D-Leu-Ala-Tyr-Pro-Arg-D-Leu-NH2 were found to show strong potentiating and relaxing activities, though they were less potent than the Mytilus SCP.

Penostatins A ( 1 ), B ( 2 ), C ( 3 ) and ( D ) have been isolated from a strain of Penicillium sp. originally separated from the marine alga Enteromorpha intestinalis , and their stereostructures have been established on the basis of spectral analyses. The compounds 1∼3 exhibited significant cytotoxicity against cultured P388 cells.

Penostatins F–I have been isolated from a strain of Penicillium sp. originally separated from the marine alga Enteromorpha intestinalis, and their stereostructures have been established on the basis of spectral analyses. All the compounds exhibit significant cytotoxicity against cultured P388 cells.

A novel violet pigment, rosacyanin B, was isolated from the petals of Rosa hyb rida cv. ‘M'me Violet’, and its structure was elucidated by spectroscopic methods. rosacyanin B is the first C-4 substituted anthocyanidin isolated from intact plants.

Two new galloylated monoterpene glycosides, 4-O-galloylalbiflorin and 4'-O-galloylpaeoniflorin, were isolated from the roots of Paeonia lactiflora that had been grown and processed in Nara prefecture, Japan. Their structures were elucidated based on spectroscopic analysis. These compounds showed androgen receptor (AR) binding activity.

Two novel triterpenoids with an unusual 14-carboxyl group, penasterone [1] and acetylpenasterol [2], were isolated from the Okinawan marine sponge Penares incrustans. The structures of 1 and 2 were established mainly on the basis of nmr spectroscopic data. The relative stereochemistry of 1 was confirmed by single-crystal X-ray diffraction analysis. Compounds 1 and 2 potently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE in a dose-dependent manner.

1. An FMRFamide-related decapeptide isolated from the anterior byssus retractor muscle (ABRM) of the bivalve mollusc, Mytilus edulis, was shown to have D-Leu as the second amino acid residue. 2. The excitatory effects of the peptide (Mytilus-FFRFamide) on the ABRM were not changed appreciably by substituting an L-Leu residue for the D-Leu residue.

Two new sesquiterpenoids, 4-methylazulene-1-carbaldehyde and 4-methylazulene-1-carboxylic acid, were isolated from cultures of Calypogeia azurea . Their structures were established by spectroscopic analysis. The sodium salt of 4-methylazulene-1-carboxylic acid was a naturally occurring water-soluble azulene that displayed anti-inflammatory and anti-ulcer activity.

A novel pentapeptide containing a D-amino acid residue was purified from the central ganglia of the African giant snail Achatina fulica Ferussac, and it was named fulicin. The primary structure of the peptide was determined to be Phe- d -Asn-Glu-Phe-Val-NH2. Fulicin potentiated tetanic contraction of the penis retractor muscle of this snail at very low concentrations, and also showed modulatory actions on the activity of the buccal and ventricular muscles and the central ganglionic neurons.

Achatin-I previously purified from the ganglia of the African giant snail Achatina fulica was isolated from the atria of this snail. Achatin-I appeared to enhance the cardiac activity in two ways; centrally this peptide increased impulse frequency and produced spike broadening of the identified heart excitatory neuron, PON, and peripherally it enhanced amplitude and frequency of the heart beat. Achatin-I showed excitatory actions not only on the heart but on several other muscles.

APGWamide (L-Ala-L-Pro-Gly-L-Trp-NH2) was purified from the ganglia of an African giant snail (Achatina fulica Ferussac). This peptide inhibited (hyperpolarized) more than half of the Achatina neurone types tested. This produced an outward current with the membrane conductance increase of RAPN (right anterior pallial neurone) under voltage clamp. The ED50 of the peptide was 6.2 x 10(-6) M (95% confidence limit: 5.0-7.8 x 10(-6) M) and the Emax was 3.9 +/- 0.2 nA. The effects were due to a membrane permeability increase to K+. The peptide is proposed as an inhibitory neurotransmitter of the Achatina neurones.

1. Effects of the molluscan neuropeptide APGW-amide and related compounds (the crustacean hormone RPCH, FAPGW-amide, PGW-amide, GW-amide and W-amide) were examined in several kinds of molluscan muscles. 2. All the compounds, except W-amide, showed qualitatively similar modulatory effects on contraction or relaxation of the muscles. The potency order of the compounds was found to be GW-amide greater than or equal to APGW-amide greater than FAPGW-amide greater than RPCH greater than PGW-amide. W-amide showed little or no effect even at 10(-4) M. 3. In the ABRM of Mytilus and the radula retractor muscle of Rapana, the active peptides were suggested to exhibit their modulatory effects by acting on the presynaptic sites in the muscles. 4. Nineteen GW-amide analogues, such as Gly-Trp-OH, Gly-Phe-NH2, D-Ala-Trp-NH2 and N-Gly-tryptamine, were also tested on some of the muscles, but all of them showed little or no effect.

The structure-activity relationships of achatin-I, a neuroactive peptide containing a D-phenylalanine residue, for producing excitatory effects on three different types of Achatina neurons, PON, TAN and d-RCDN, were studied under the voltage clamp method. Of the peptides examined, only Gly-Gly-D-Phe-L-Ala-L-Asp (IV), D-Phe-L-Ala-L-Asp (V) and Gly-D-Phe-L-Ala-L-Asn (XVI) produced an inward current with increased membrane conductance similar to achatin-I (I). The structure-activity relationship was essentially the same for the three Achatina neuron types. The equiactive molar ratios (EMRs) of the active peptides vs. achatin-I (I) were calculated from their dose-response curves: 8 – 60 for Gly-Gly-DPhe-L-Ala-L-Asp (IV), 200 - >250 for D-Phe-L-Ala-L-Asp (V) and >200 for Gly-D-Phe-L-Ala-L-Asn (XVI). These values indicate that the achatin-I receptor in the Achatina neurons is highly structure-specific.

In graminaceous plants such as barley, oats, and wheat, novel iron-chelating amino acids are secreted from the roots. A typical example is mugineic acid. A phytosiderophore, mugineic acid significantly stimulates iron-uptake and chlorophyll synthesis in rice plants. Most microbial siderophores have hydroxamate or phenolate groups as Fe(III)-coordination donors, while phytosiderophores consist of carboxyl, amine, and hydroxyl groups as the ligand functional groups. The mugineic acid-Fe(III) complex and its structurally analogous Co(III) complex have been characterized by some spectroscopic and X-ray diffraction methods. The coordination of mugineic acid to Co(III) and Fe(III) ions involves the azetidine nitrogen, secondary amine nitrogen, both terminal carboxylate oxygens as basal planar donors, and the hydroxyl oxygen and intermediate carboxylate oxygen as axial donors in nearly octahedral configuration. The Mossbauer (ΔEQ = 0.24 and δ Fe = + 0.39 mm/sec) and ESR (g = 9.4, 4.51, 4.44, and 4.31) parameters of the mugineic acid-Fe(III) complex are characteristic of high-spin (S = 5/2) ferric type. Of special interest is the apparent high reduction potential (E1/2 = − 102 mV vs. NHE) of the mugineic acid-Fe(III) complex, as compared to those of the microbial hydroxamates and ferric enterobactin. The mechanism of iron-absorption and -transport in gramineous plants probably includes Fe(III)-solubilization by mugineic acid and reduction from the thermodynamically stable ferric mugineic acid complex (log K ML M = 18.1) to the weakly bound ferrous complex (log K ML M = 8.1).

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)-phytosiderophore complexes. We identified a gene specifically encoding an iron-phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)-phytosiderophore transporter in maize. Real-time RT-PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron-deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti-HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)-MA, but not iron-nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)-phytosiderophore involved in primary iron acquisition from soil in barley roots.