Nectriatide, a Potentiator of Amphotericin B Activity from Nectriaceae sp. BF-0114.

A new compound, designated nectriatide (1), was isolated as a potentiator of amphotericin B (AmB) activity against Candida albicans from the culture broth of Nectriaceae sp. BF-0114. This structure was elucidated based on spectroscopic analyses (1D and 2D NMR data), chemical methods, and total synthesis. Compound 1 was a unique cyclotetrapeptide consisting of l-N-methyltyrosine, anthranilic acid, l-alanine, and l-valine. Compound 1 and several synthetic derivatives, including linear peptides, potentiated AmB activity against C. albicans by up to 16-fold (the MIC value of AmB decreased from 0.5 µg/mL to 0.031 µg/mL in combination with test compound).

Stereoisomer-Specific Induction of G2/M Phase Arrest and Apoptosis by 9-(E,Z)-Hydroxyoctadecadienoic Acid in Mouse Lymphoma Cells.

Hydroxyoctadecadienoic acids (HODEs) are generated by oxidation of linoleic acid in vivo and thought to mediate various pathophysiological responses. In this study, we examined the effects of HODEs on EL4 mouse lymphoma cell growth and found that 9-(E,Z)-HODE inhibited EL4 cell growth in a dose-dependent manner, whereas no such growth inhibition was observed with other isomers (9-(E,E)-, 13-(Z,E)-, or 13-(E,E)-HODE), suggesting that the growth-inhibitory effect of HODEs was stereospecific. Analysis by flow cytometry (FACS) with annexin V and propidium iodide (PI) staining showed that 9-(E,Z)-HODE induced apoptosis with G2/M phase arrest. We next examined the growth inhibition profile of 9-(E,Z)-HODE against a panel of 39 human cancer cell lines (JFCR39). The fingerprint of growth inhibition by 9-(E,Z)-HODE exhibited a high degree of similarity to that by MLN4924, an inhibitor of NEDD8-activating enzyme. The intracellular NEDD8 (ubiquitin-like protein) expression in EL4 cells was decreased by the treatment with 9-(E,Z)-HODE as assessed by immunoblotting and flow cytometry. In conclusion, 9-(E,Z)-HODE specifically induced G2/M phase arrest and apoptosis, and the decrease of NEDD8 expression might be involved in this effect.

New Mastoparan Peptides in the Venom of the Solitary Eumenine Wasp Eumenes micado.

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic α-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly α-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes.

New mastoparan peptides in the venom of the solitary eumenine wasp eumenes micado

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic a-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly a-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes

New mastoparan peptides in the venom of the solitary eumenine wasp eumenes micado

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic a-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly a-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes

Degradation of Jatropha curcas phorbol esters derived from Jatropha oil cake and their tumor-promoting activity.

Large amount of oil cake is generated during biodiesel production from Jatropha seeds. Although Jatropha oil cake is rich in plant nutrients, presence of toxic phorbol esters restricts the usage of oil cake as a fertilizer. The objective of this study is to evaluate the components and tumor promoting activity of phorbol esters in Jatropha oil cake-supplemented soil and plants grown in the treated soil. Contents and their biological activity of Jatropha phorbol esters in soil and plants were sequentially analyzed by high-performance liquid chromatography (HPLC) and in vitro cell transformation assay, respectively. Disappearance of Jatropha phorbol-ester-specific peaks were followed with HPLC during incubation of Jatropha oil cake with soil for five weeks. Along with the degradation of Jatropha phorbol ester in soil, tumor-promoting activity in the sample was also attenuated and ultimately disappeared. Jatropha phorbol esters and tumor promoting activity were not detected from mustard spinach grown in the Jatropha oil cake-supplemented soil. In addition, the esterase KM109 degrades DHPB (see definition below; Jatropha phorbol ester) and reduced its tumor-promoting activity. From these data, we conclude: (1) components and tumor promoting activity of Jatropha phorbol esters in the oil cake disappeared completely by incubation with soil for five-week, (2) Jatropha phorbol esters did not transfer into plants grown in the Jatropha oil cake-supplemented soil, and (3) DHPB can be degraded by esterase from soil bacterium. These observations are useful for utilization of Jatropha oil cake as a fertilizer.

New dinapinone derivatives, potent inhibitors of triacylglycerol synthesis in mammalian cells, produced by Talaromyces pinophilus FKI-3864.

Eight new dinapinones, AB1, AB2, AC1, AC2, AD1, AD2, AE1 and AE2, were isolated from the culture broth of Talaromyces pinophilus FKI-3864. The structures of these dinapinones were elucidated by various NMR experiments. All these dinapinones possessed the same biaryl dihydronaphthopyranone skeleton consisting of a heterodimer with one monapinone A and one different monapinone. Dinapinones AB1 and AB2, consisting of monapinones A and B, were atropisomers. Similarly, dinapinones AC1 and AC2, consisting of monapinones A and C, dinapinones AD1 and AD2, consisting of monapinones A and D, and dinapinones AE1 and AE2, consisting of monapinones A and E, were atropisomers. Dinapinone AB2 showed potent inhibition of triacylglycerol (TG) synthesis in intact mammalian cells with an IC50 value of 1.17 µM, whereas the other dinapinones showed weak inhibition of TG synthesis.

Characterization of peptides obtained from digests of bovine brain which accelerate structural conversions of the recombinant bovine prion protein.

Structural changes of proteins are thought to involve specific protein-peptide interactions, thus we hypothesize that certain peptides may contribute to the conformational change of prion proteins. Hence peptide libraries were constructed from partial digests of bovine brain. Using a recently developed conversion assay method, we have screened peptides responsible for structural conversion. Positive components were identified of which amino acid sequences were elucidated by top-down sequencing using mass spectrometry. A database search identified a peptide derived from synaptophysin. This peptide was chemically synthesized to confirm acceleration of the structural change of recombinant bovine prion protein.

Isolation and identification of C-19 fatty acids with anti-tumor activity from the spores of Ganoderma lucidum (reishi mushroom).

We previously showed that ethanolic extracts of spores of Ganoderma lucidum inhibit tumor cell proliferation and induce apoptosis of HL-60 cells. The active constituents appeared to be long-chain fatty acids, particularly carbon-19 (C-19) fatty acids which have not been reported in spores of Ganoderma lucidum. In the present study, two of these C-19 fatty acids which are key compounds in the activities, were identified as their 2-naphthyl ester derivatives after esterification of a mixture of fatty acids obtained from the spores. The active compounds were determines as nonadecanoic acid and cis-9-nonadecenoic acid. The location of the double bond of cis-9-nonadecenoic acid was demonstrated by GC-MS analysis, based on the fragmentation pattern of the adduct prepared from the fatty acid and dimethyl disulfide.

Characterization of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use.

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.

Structure and total synthesis of fungal calpinactam, a new antimycobacterial agent.

A new fungal metabolite designated calpinactam (1) was isolated from the culture broth of Mortierella alpina FKI-4905, and its structure was elucidated by spectroscopic analyses including NMR experiments. Calpinactam was found to be a hexapeptide with a caprolactam ring at its C-terminal. Its absolute stereochemistry was determined by amino acid analysis and total synthesis. Calpinactam selectively inhibited the growth of mycobacteria among various microorganisms. The MIC values of calpinactam against Mycobacterium smegmatis and M. tuberculosis were 0.78 and 12.5 microg/mL, respectively.

Possible involvement of long chain fatty acids in the spores of Ganoderma lucidum (Reishi Houshi) to its anti-tumor activity.

During our isolation of biologically active substances from the spores of Ganoderma lucidum (Reishi Houshi, G. lucidum) guided by the inhibitory activity on HL-60 cell proliferation, NMR spectroscopic and mass spectrometric data indicate the substance contains a mixture of several long chain fatty acids. Hence, in this study, we have examined the inhibitory effects of an ethanolic extract of the spores of G. lucidum as the spore extract, on the proliferation of various human cancer cell lines by comparison with several authentic long chain fatty acids. Of the fatty acids we examined nonadecanoic acid (C19:0) showed the highest inhibitory activity for HL-60 cell proliferation with IC(50) values of 68+/-7 microM followed by heptadecanoic acid (C17:0, 120+/-23 microM), octa- (C18:0, 127+/-4 microM) and hexadecanoic acids (C16:0, 132+/-25 microM), respectively. The corresponding unsaturated fatty acids containing one double bond such as cis-10-nonadecenoic acid (C19:1), cis-9-octadecenoic acid (C18:1), cis-10-heptadecenoic acid (C17:1) and cis-9-hexadecenoic acid (C16:1) were less effective. The ethanolic extract of spores of G. lucidum were shown by annexin-V FITC/PI double staining to induce apoptosis of HL-60 cells in a similar way to cis-10-nonadecenoic acid (C19:1). These unsaturated fatty acids probably inhibit tumor necrosis factor production induced by lipopolysaccharide in a mouse macrophage preparation. Our results suggest the spores of G. lucidum contain 19-carbon fatty acids as one of the components for characteristics of its physiological effects.

Absorption and tissue distribution of an immunomodulatory alpha-D-glucan after oral administration of Tricholoma matsutake.

Alpha-D-glucan (MPG-1) separated from Tricholoma matsutake (CM6271) has been reported to show immunomodulatory activities. In this study, the plasma concentration and tissue distribution of MPG-1 after CM6271 oral administration were investigated as part of the action mechanism analysis. When CM6271 was orally administered in a single dose to mice, MPG-1 was absorbed via the intestinal tract, appeared in plasma after 16 h, was gradually excreted from the blood, and fell to background level after 48 h. The time course analysis of MPG-1 in plasma showed the following pharmacokinetic parameters of MPG-1: tmax = 24 h; Cmax = 161.1 ng/mL; AUC(0-infinity) = 2559.7 ng x h/mL. Moreover, MPG-1 was confirmed to localize in Peyer's patches, mesenteric lymph nodes (MLN), and the spleen and to promote IL-12 p70 production and NK cell activity. These results suggest that MPG-1 stimulated the intestinal immune system through Peyer's patches; moreover, it was taken into the blood and stimulated the systemic immune system.

Development of an enzyme-linked immunosorbent assay to detect an immunomodulatory alpha-D-glucan-protein complex, MPG-1, in basidiomycete Tricholoma matsutake and related processed foods.

We previously isolated a novel immunomodulatory alpha-(1,4)(1,6)(1,2)- d-glucan-protein complex (MPG-1) from mycelia of Tricholoma matsutake in basidiomycetes. In the present study, we raised a polyclonal antibody by immunizing rabbits with MPG-1 and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) system to examine the distribution of MPG-1 among edible mushrooms and related processed foods. The system detected MPG-1 quantitatively at concentrations of more than 10 ng/mL, with a coefficient of variation of less than 10% by intra-assay and interassay precision. Analysis with the system of chemically modified MPG-1 suggested that the sugar moiety was mainly involved in the detection. The system detected MPG-1 in the extracts of the fruiting bodies of T. matsutake but not in those of 34 other basidiomycete species. Moreover, a significant amount of MPG-1 was detected in the extracts of their cultured mycelia. The antigenic structure of MPG-1 was relatively stable in terms of pH and temperature. MPG-1 was detected in processed foods supplemented with T. matsutake. These results suggest that MPG-1 is distributed predominantly in T. matsutake species and that the ELISA system can detect it in processed foods supplemented with T. matsutake.

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

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

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

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

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes.

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.

A study of cross-reactions between mango contact allergens and urushiol.

The allergens causing mango dermatitis have long been suspected to be alk(en)yl catechols and/or alk(en)yl resorcinols on the basis of observed cross-sensitivity reactions to mango in patients known to be sensitive to poison ivy and oak (Toxicodendron spp.). Earlier, we reported the 3 resorcinol derivatives: heptadecadienylresorcinol (I), heptadecenylresorcinol (II) and pentadecylresorcinol (III); collectively named 'mangol', as mango allergens. In this study, we extracted the 1st 2 components (I and II) from the Philippine mango, adjusted them to 0.05% concentration in petrolatum and patch tested the components on 2 subjects with mango dermatitis. Both subjects reacted to I. 1 subject also elicited a weaker positive reaction to II. To investigate the cross-reaction between mangol and urushiol, we also patch tested the same subjects with urushiol. The subject sensitive to II reacted to urushiol. 6 subjects with a history of lacquer contact dermatitis and positive reactions to urushiol were similarly patch tested. 5 persons reacted to I. 2 subjects also exhibited a slower but positive reaction to II. This is the 1st report in which heptadec(adi)enyl resorcinols known to be present in mango have been shown to elicit positive patch test reactions in mango-sensitive patients.

Anti-allergic substances contained in the pollen of Cryptomeria japonica possess diverse effects on the degranulation of RBL-2H3 cells.

Following prolonged exposure to some of the flavonoids with RBL-2H3 cells, secretion of hexosaminidase, a granule constituent, stimulated by an immunologic was enhanced. RBL-2H3 cells do not normally respond to polybasic secretagogues, but as reported here, they do so after prolonged exposure. Effect of flavonoids on secretion of hexosaminidase was also investigated. Of the thirteen flavonoids, quercetin and fisetin were the most potent inhibitors. A structure-activity study indicated that the position, number, and substitution of the hydroxy group of the B ring and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of secretary granules in RBL-2H3 cells.

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry.

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.