Non-Enzymatic Oxidation of a Pentagalloylglucose Analogue into Members of the Ellagitannin Family.

The occurrence of more than 1000 structurally diverse ellagitannins has been hypothesized to begin with the oxidation of penta-O-galloyl-ß-d-glucose (ß-PGG) for the coupling of the galloyl groups. However, the non-enzymatic behavior of ß-PGG in the oxidation is unknown. Disclosed herein is which galloyl groups tended to couple and which axial chirality was predominant in the derived hexahydroxydiphenoyl groups when an analogue of ß-PGG was subjected to oxidation. The galloyl groups coupled in the following order: at the 4,6-, 1,6-, 1,2-, 2,3-, and 3,6-positions with respective S-, S-, R-, S-, and R-axial chirality. Among them, the most preferred 4,6-coupling reflected the what was observed for natural ellagitannins. A new finding was that the second best coupling occured at the 1,6-positions. With the detection of a 3,6-coupled product, this work demonstrated that even ellagitannin skeletons with an axial-rich glucose core may be generated non-enzymatically.

Transgenic petunia with the iron(III)-phytosiderophore transporter gene acquires tolerance to iron deficiency in alkaline environments.

Iron is an essential nutrient for all plants. However, terrestrial plants often suffer from iron deficiency in alkaline soil due to its extremely low solubility. Alkaline soil accounts for about 30% of all cultivated ground in the world. Plants have evolved two distinct strategies, I and II, for iron uptake from the soil. Dicots and non-graminaceous monocots use Strategy I, which is primarily based on the reduction of iron(III) to iron(II) and the uptake of iron(II) by the iron-regulated transporter, IRT1. In contrast, graminaceous plants use Strategy II to efficiently acquire insoluble iron(III). Strategy II comprises the synthesis and secretion of iron-chelating phytosiderophores, such as mugineic acids and the Yellow Stripe 1 transporter proteins of the iron(III)-phytosiderophore complex. Barley, which exhibits the highest tolerance to iron deficiency in alkaline soil among graminaceous plants, utilizes mugineic acids and the specific iron(III)-mugineic acids transporter, HvYS1. In this study, we established the transgenic plant Petunia hybrida, which originally had only Strategy I, by introducing the HvYS1 transporter gene derived from barley. When the transgenic plants were grown hydroponically in media containing the iron(III)-2'-deoxymugineic acid complex, free 2'-deoxymugineic acid and its iron(III) complex were detected in the root extract of the transgenic plant by electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry. The growth of the transgenic petunia was significantly better than that of the control host in alkaline conditions. Consequently, the transgenic plant acquired a significantly enhanced tolerance to alkaline hydroponic media in the presence of the iron(III)-2'-deoxymugineic acid complex. Furthermore, the flower color of the transgenic plant deepened. The results showed that iron-phytosiderophore complexes and their transporters can potentially be utilized to overcome the worldwide iron uptake problems to diverse plant species that are found in areas with alkaline conditions.

Physiological characterization of aluminum tolerance and accumulation in tartary and wild buckwheat.

Ionic aluminum (Al) is toxic for plant growth, but some plant species are able to accumulate Al at high concentrations without showing toxicity symptoms. In order to determine whether other species in the genus Fagopyrum are able to accumulate Al like common buckwheat (Fagopyrum esculentum), we investigated the external and internal detoxification mechanisms of Al in two self-compatible species: tartary (Fagopyrum tataricum) and wild buckwheat (Fagopyrum homotropicum). Both tartary and wild buckwheat showed high Al tolerance comparable to common buckwheat. Furthermore, these two species also secreted oxalate rapidly from the roots in response to Al in a time-dependent manner. Both tartary and wild buckwheat accumulated > 1 mg g(-1) Al in the leaves after short-term exposure to Al. Analysis with (27) Al-nuclear magnetic resonance (NMR) revealed that Al was present in the form of Al-oxalate (1 : 3 ratio) in the roots and leaves, but in the form of Al-citrate (1 : 1 ratio) in the xylem sap in both species. These results indicate that similar to common buckwheat, both tartary and wild buckwheat detoxify Al externally and internally, respectively, by secreting oxalate from the roots and by forming the Al-oxalate complex, which is a nonphytotoxic form. These features of Al response and accumulation may be conserved in genus Fagopyrum.

Penasins A-E, long-chain cytotoxic sphingoid bases, from a marine sponge Penares sp.

Five sphingoid bases, penasin A (1), penasin B (2), and a mixture of penasins C-E (3-5), were identified from a marine sponge Penares sp. as cytotoxic constituents. The structure of the common polar head part was assigned by analysis of the NMR data, whereas the structures of the long aliphatic chains including the locations of double bond(s) and a branched methyl group were determined by analysis of tandem FABMS and (13)C NMR data together with the GC-MS analysis of ozonolysis products. The absolute configuration of the headgroup was defined for the mixture of 3-5 by the modified Mosher method. Penasins exhibit moderate cytotoxicity against HeLa and P388 cells.

Alchivemycin A, a bioactive polycyclic polyketide with an unprecedented skeleton from Streptomyces sp.

Alchivemycin A, a novel polycyclic polyketide, was isolated from the culture extract of a plant-derived actinomycete Streptomyces sp. The structure and relative configuration were elucidated by spectroscopic analysis and X-ray crystallography, and the absolute configuration was determined by a (1)H NMR anisotropy method using MPA ester derivatization. The new compound contains an unprecedented heterocyclic ring system, 2H-tetrahydro-4,6-dioxo-1,2-oxazine. Alchivemycin A showed potent antimicrobial activity against Micrococcus luteus and inhibitory effects on tumor cell invasion.

Androgen modulators from the roots of Paeonia lactiflora (paeoniae radix) grown and processed in nara prefecture, Japan.

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.

Inconspicamide, new N-acylated serinol from the marine sponge Stelletta inconspicua.

A new N-acylated serinol, inconspicamide (1), was isolated from the marine sponge, Stelletta inconspicua, together with a glyceryl ether (2). Their structures were determined on the basis of spectroscopic data and the modified Mosher analysis. They exhibited moderate cytotoxic activity against HeLa human cervical cancer cells.

A prodigiosin analogue inactivates NADPH oxidase in macrophage cells by inhibiting assembly of p47phox and Rac.

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.

Cytotoxic C47-polyacetylene carboxylic acids from a marine sponge Pertrosia sp.

Two new polyacetylene carboxylic acids, petroformynic acids B (3) and C (4), were isolated from a marine sponge Pertrosia sp. as cytotoxic constituents. Their structures were determined by interpretation of 2D NMR data and tandem FABMS data. Absolute stereochemistry of 3 was assigned by application of the modified Mosher analysis. Petroformynic acids exhibit moderate cytotoxic activity against P388 cells.

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors.

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.

A specific transporter for iron(III)-phytosiderophore in barley roots.

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.

Induction of morphological changes in model lipid membranes and the mechanism of membrane disruption by a large scorpion-derived pore-forming peptide.

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct alpha-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10(-3) s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at approximately 30 degrees to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro.

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis.

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.

Solution structure of IsTX. A male scorpion toxin from Opisthacanthus madagascariensis (Ischnuridae).

The novel sex-specific potassium channel inhibitor IsTX, a 41-residue peptide, was isolated from the venom of male Opisthacanthus madagascariensis. Two-dimensional NMR techniques revealed that the structure of IsTX contains a cysteine-stabilized alpha/beta-fold. IsTX is classified, based on its sequential and structural similarity, in the scorpion short toxin family alpha-KTx6. The alpha-KTx6 family contains a single alpha-helix and two beta-strands connected by four disulfide bridges and binds to voltage-gated K(+) channels and apamin-sensitive Ca(2+)-activated K(+) channels. The three-dimensional structure of IsTX is similar to that of Heterometrus spinifer toxin (HsTX1). HsTX1 blocks the Kv1.3 channel at picomolar concentrations, whereas IsTX has much lower affinities (10 000-fold). To investigate the structure-activity relationship, the geometry of sidechains and electrostatic surface potential maps were compared with HsTX1. As a result of the comparison of the primary structures, Lys27 of IsTX was conserved at the same position in HsTX1. The analogous Lys23 of HsTX1, the most critical residue for binding to potassium channels, binds to the channel pore. However, IsTX has fewer basic residues to interact with acidic channel surfaces than HsTX1. MALDI-TOF MS analysis clearly indicated that IsTX was found in male scorpion venom, but not in female. This is the first report that scorpion venom contains sex-specific compounds.

Orientation and pore-forming mechanism of a scorpion pore-forming peptide bound to magnetically oriented lipid bilayers.

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly3, Gly8, Leu12, Phe17, or Ser18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala4, Gly8, and Ala9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.

Designing analogues of mini atrial natriuretic peptide based on structural analysis by NMR and restrained molecular dynamics.

Analogues of mini atrial natriuretic peptide (miniANP) that provide conformational properties related to biological activity were designed on the basis of the structure revealed by NMR and restrained molecular dynamics (rMD) simulation, and an analogue with a high level of biological activity was successfully obtained. MiniANP is a cyclic pentadecapeptide analogue of atrial natriuretic polypeptide (ANP). The conformation of miniANP analyzed by NMR and rMD simulation indicated that positive phi angles are preferred for Gly(5) and Gly(6), which is typical for D-amino acids. On the basis of the structural information, [D-Ala(5)]miniANP, [D-Ala(6)]miniANP, and [D-Ala(5) D-Ala(6)]miniANP were synthesized. The biological activity of [D-Ala(5)]miniANP was stronger than that of miniANP, confirming that Gly(5) of miniANP takes a positive phi angle on binding to the receptor. Conformational analysis of these analogue peptides by NMR suggested that a turnlike conformation at residues 6-9 and a proximate pair formed by side chains of Phe(4) and Ile(11) are important for the biological activity.