Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl ß-methoxyacrylate group and a strobilurin analogue bearing modified ß-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Investigation of the Protection of the C4 Hydroxyl Group in Macrobicyclic Kdo Donors.

Chemical synthesis of 3-deoxy-d-manno-2-octulosonic acid (Kdo)-containing glycans, such as bacterial lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs), is in high demand for the development of vaccines against pathogenic bacteria. We have recently achieved the complete α-stereoselective glycosidation of Kdo using a macrobicyclic donor tethered at the C1 and C5 positions. In this study, to expand the scope of Kdo glycosidation, we sought to protect the 4-OH group, thereby shortening the reaction time and ensuring the conversion of the glycosyl acceptor via its selective removal. The protection of the 4-OH group influenced the reactivity of the Kdo donor, and the triisopropylsilyl (TIPS) group acted as a selectively removable booster. The 4-O-TIPS donor allowed the synthesis of the α(2,4)-linked dimeric Kdo sequence, which is widely found in bacterial LPSs.

5-hydroxymethyl-2-furaldehyde purified from Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice concentrate inhibits melanogenesis in B16 mouse melanoma cells.

Pear juice concentrate prepared by boiling Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice can significantly inhibit the activity of tyrosinase, a key enzyme in melanin synthesis in human skin. Using the ethanol extract of pear juice concentrate, we homogeneously purified an active compound that was identified as 5-hydroxymethyl-2-furaldehyde (5-HMF) through 1H- and 13C-NMR and mass spectroscopy. We observed that 5-HMF inhibited the monophenolase and diphenolase activities of mushroom tyrosinase as a mixed-type inhibitor (K i values of 3.81 and 3.70 mmol/L, respectively). In B16 mouse melanoma cells, treatment with 170 µmol/L of 5-HMF significantly reduced α-melanocyte-stimulated melanin synthesis by suppressing the cyclic adenosine monophosphate-dependent signaling pathway involved in melanogenesis. The results of our study indicated that 5-HMF can be potentially used as a skin-lightening agent in the cosmetic industry. Abbreviations: AC: adenylate cyclase; CREB: cAMP response element-binding protein; dhFAME: S-(-)-10,11-Dihydroxyfarnesoic acid methyl ester; DMEM: dulbecco's modified eagle medium; l-DOPA: 3-(3,4-Dihydroxyphenyl)- l-alanine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HEPES: 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid; 5-HMF: 5-Hydroxymethyl-2-furaldehyde; MITF: microphthalmia-associated transcription factor; α-MSH: α-Melanocyte-stimulating hormone; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SDS: sodium dodecyl sulfate; TRP1: tyrosinase-related protein 1; TRP2: tyrosinase-related protein 2.

A lemon myrtle extract inhibits glucosyltransferases activity of Streptococcus mutans.

Streptococcus mutans is a bacterium found in human oral biofilms (dental plaques) that is associated with the development of dental caries. Glucosyltransferases (GTFs) are key enzymes involved in dental plaque formation, and compounds that inhibit their activities may prevent dental caries. We developed a screening system for GTF-inhibitory activities, and used it to profile 44 types of herbal tea extracts. Lemon myrtle (Backhousia citriodora) extract exhibited the highest GTF-inhibitory activity, with an IC50 for GTF in solution of 0.14 mg mL-1. Furthermore, lemon myrtle extracts had the third-highest polyphenol content of all tested extracts, and strongly inhibited S. mutans biofilm. Interestingly, lemon myrtle extracts did not inhibit cell growth.

Convergent synthesis of 4,5-branched inner-core oligosaccharides of lipopoly- and lipooligosaccharides.

The convergent synthesis of branched inner-core oligosaccharides of lipopoly- and lipooligosaccharide with a 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) disaccharide acceptor was achieved. The l-glycero-d-manno-heptopyranose (Hep) units for the branched core oligosaccharide Galß(1-4)Glcß(1-4)Hep and Hepα(1-3)Hep were prepared from the corresponding Hep building blocks. To obtain 4,5-branched core oligosaccharide structures, the common acceptor Kdoα(2-4)Kdo was glycosylated with the Hep units.

Biosynthesis of integric acid isolated from the wood-decay fungus Xylaria feejeensis 2FB-PPM08M.

The biosynthesis of integric acid, a secondary metabolite of the wood-decay fungus Xylaria feejeensis strain 2FB-PPM08M, has been studied. Labeling experiments using [1-(13)C], [2-(13)C] and [1,2-(13)C2] acetate and L-methionine (methyl-(13)C) were separately performed with fungal culture. The labeling patterns of these metabolites indicated the same origin, and determined that integric acid was formed through the condensation of a sesquiterpene and a polyketide. These experiments showed that side chain of compounds would be synthesized by the polyketide pathway, while the ring carbon indicated the biosynthesis of compounds via the mevalonate pathway.

Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

Identification of Cyclocybe erebia metabolites that affect the circadian rhythm of Eluc expression under control of Bmal1 promoter in mouse fibroblast cells.

Pharmacological intervention of circadian rhythms is a potentially useful approach for ameliorating various health problems caused by disturbed circadian rhythms including sleep disorder and metabolic diseases. To find compounds that affect circadian rhythms, we screened mushroom extracts using mouse cells expressing the luciferase gene under the control of the mouse Bmal1 promoter. The culture filtrate extract from the basidiomycete Cyclocybe erebia enhanced the oscillation of bioluminescence caused by the expression of the luciferase gene and prolonged the period of bioluminescence. Bioassay-guided fractionation of the extract resulted in purification of compounds 1 and 2. Spectroscopic analyses along with single-crystal X-ray diffraction analysis, revealed that these compounds were diterpenoids with a unique skeleton and a fused ring system comprising 3-, 7-, and 5-membered rings. Compounds 1 and 2 were named cyclocircadins A and B, respectively. These findings suggested that natural diterpenoids could be a source of compounds with the activity affecting circadian rhythms.

Multiple effects of gymnopilin on circulatory system of the rat.

Gymnopilin is one of the substances produced by the hallucinogenic mushroom, Gymnopilus junonius. In this study, we examined effects of gymnopilins purified from wild fruiting bodies of G. junonius on contractile activity of aorta preparations and blood pressure in rats. Gymnopilins at lower concentrations than 5 mg/mL did not evoke contraction of helical strips of the thoracic aorta. In contrast, gymnopilins (5 mg/mL) applied to the aorta strips pre-contracted by norepinephrine (100 nM) caused relaxation. This relaxing action did not depend on the activity of the endothelium cells. The relaxing effect of 5-mg/mL gymnopilins was observed in aorta strips contracted by angiotensin II (10 nM) and the high K+ solution (60 mM). Moreover, the adenylyl cyclase inhibitor, SQ-22536, significantly inhibited the relaxing effect of gymnopilins at 1 mg/mL on the norepinephrine-contracted strips. These results suggested that gymnopilins acted directly on smooth muscle cells of the aorta and activated the cAMP-dependent cascade to cause the vasodilation. Paradoxically, gymnopilins injection into the jugular vein transiently increased blood pressure without affecting the heart rate. This result suggests that gymnopilins increase cardiac output and/or tension of the artery through the excitation of the vasomotor nerve that overcame the direct relaxing effect on the vascular smooth muscle.

Western blot analysis of neisserial lipooligosaccharide at the lower femtomole level with normal human sera.

Lipooligosaccharide (LOS) is a major immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae. Recent immunochemical studies have found that normal human sera (NHS) contain bactericidal anti-LOS antibodies that bind to the oligosaccharide (OS) moiety of neisserial LOS. Although affinity-purified anti-LOS antibodies can be characterized using 10-100 ng of LOS samples (up to a few tens of pmoles), a more sensitive immunoblotting assay must be established in order to analyze NHS directly and characterize anti-LOS antibodies without affinity purification. We examined analytical PAGE/blot conditions using a 15-well mini gel. For the first time, Western blot detection of LOS at the lower femtomole level was accomplished by both chromogenic and chemiluminescent detection. A model LOS, 15253 LOS, was detected in a low femtomole range (62.5-500 pg, 16-125 femtomole) even with 10 pM of a monoclonal antibody (MAb) 2C7. Furthermore, detection of similar amounts (50-250 femtomole) of neisserial LOSs and Salmonella truncated lipopolysaccharides (LPSs) was also possible with 1:50 and with 1:100 diluted NHS. The results obtained here indicate that the binding of IgG in NHS to the LOS and LPS samples is probably due to their carbohydrate moieties. The detection level accomplished in this study should help not only to further characterize anti-LOS antibodies in blood and body fluids but also to analyze carbohydrate structures that are recognized by them.

Full Stereocontrol in α-Glycosidation of 3-Deoxy-d-manno-2-octulosonic Acid (Kdo) Using Macrobicyclic Glycosyl Donors.

We describe a method for the α-selective glycosidation of 3-deoxy-d-manno-2-octulosonic acid (Kdo) using a macrobicyclic Kdo donor as the precursor of a bridgehead oxocarbenium ion, whose stereoselectivity is not affected by the substrate structure and reaction conditions. Strapping Kdo via tethering in the α-configuration at the C1 and C5 positions completely blocked nucleophilic attack to the ß-face of the anomeric center by sterically hindering the bicyclic system, realizing full α-selectivity during glycosidation.

Novel effects of extracts from poisonous mushrooms on expression and function of the human ether-a-go-go-related gene channel.

UNLABELLED: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr), which plays a pivotal role in cardiac action potential repolarization. Inherited mutations of this gene cause Long QT syndrome type 2. hERG expression is altered by several types of drugs as well as by temperature. Heat shock protein 70 (Hsp70) and Heat shock cognate protein 70 (Hsc70) have reciprocal effects on hERG proteins. We examined the effects of poisonous mushrooms on hERG protein expression and its channel function. METHODS: We evaluated the effects of several types of poisonous mushrooms on the expression and function of wild-type hERG by Western blotting, reverse transcription polymerase chain reaction (PCR), and patch clamping in transfected HEK293 cells and mouse HL-1 cardiomyocytes. RESULTS: Extracts of Gymnopilus junonius (junonius) increased expression of both hERG and Hsp70 in HEK293 cells with concomitant decrease in Hsc70, whereas extracts of Amanita ibotengutake (ibotengutake) decreased hERG proteins with increase in Hsc70. Knockdown of Hsp70 and Hsc70 by small interfering RNA abolished the effects of the two mushrooms on hERG, respectively. Certain fractions of junonius increased expression of hERG proteins. hERG currents were increased by extracts of junonius, resulting in shortening of action potential duration (APD). In contrast, hERG currents were decreased and APD was prolonged by extracts of ibotengutake. CONCLUSION: junonius enhanced the expression and function of hERG by increasing Hsp70 and decreasing Hsc70. Ibotengutake decreased hERG expression via increase in Hsc70. Constituents of junonius may have the potential for use in treatment of arrhythmia.

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity.

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic L-aminoacyl-pNAs and exhibited little activity toward D-Ala-pNA and D-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides--L-kyotorphin (L-Tyr-L-Arg) and D-kyotorphin (L-Tyr-D-Arg)--using L-Tyr-NH(2) and D- and L-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.

Argeloside I inhibits the pathogenicity of Porphyromonas gingivalis TDC60.

Porphyromonas gingivalis, a major pathogen associated with chronic periodontitis, produces several virulence agents in the outer cell membrane, including gingipains and hemagglutinins. These virulence factors enable the bacteria to adhere to periodontal tissue and degrade host proteins to obtain the nutrients needed for dental plaque formation. P. gingivalis TDC60 was recently identified as the most aggressive P. gingivalis strain to dates. In this study, we isolated a known pregnane glycoside, argeloside I, from the aqueous extract of Solenostemma argel leaves. Argeloside I completely hindered the growth of P. gingivalis TDC60 and inhibited the production of hemagglutinins as well as Arg- and Lys-specific gingipains. Our results demonstrate a new function of pregnane glycosides. Argeloside I may be a candidate for reducing the risk associated with P. gingivalis TDC60 and its adhesion factors.

Isolation of isolactarane sesquiterpenes from a Phlebia tremellosa culture filtrate and their growth promotion effects on lettuce roots.

The ethyl acetate extract of the culture filtrate of Phlebia tremellosa promoted elongation of the lateral roots of lettuce seedlings at 250 µg/mL. We purified two compounds that promote root elongation by using activity-guided chromatographic fractionation. On the basis of spectroscopic analyses, these compounds were identified to be isolactarane sesquiterpenes derived from the dehydrogenation of merulactone, which was previously isolated from the same species. We named the purified compounds phlelactones A and B. Phlelactones A and B promoted primary root elongation at 100-300 and 10-30 µg/mL and the elongation and formation of lateral roots at 300-1000 and 30-100 µg/mL, respectively.

Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley.

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley (Hordeum vulgare L.), we reported previously the existence of two BADH genes (BBD1 and BBD2) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K(m) of 18.9 microM and 19.9 mM, respectively. In addition, V(max)/K(m) with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of omega-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4-N-trimethylaminobutyraldehyde and 3-N-trimethylaminopropionaldehyde.

4-N-trimethylaminobutyraldehyde dehydrogenase: purification and characterization of an enzyme from Pseudomonas sp. 13CM.

4-N-trimethylaminobutyraldehyde dehydrogenase from Pseudomonas sp. 13CM was purified 14-fold to apparent homogeneity by hydrophobic chromatography on a Phenyl-Toyopearl, and affinity chromatography was done on a 5'-AMP Sepharose4B in the presence of dithiothreitol. The enzyme was found to be a trimer with identical 55 kDa subunits. The isoeletric point was found to be 5.5. The optimum temperature and pH were 40 degrees C and pH 10.0. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and analog inhibition. The K(m) values for 4-N-trimethylaminobutyraldehyde, 4-dimethylaminobutyraldehyde, and NAD(+) were 7.4, 51, and 125 microM respectively. The enzyme was inhibited by SH reagents, and by heavy metal ions.

Transcriptional regulation of two endoglucanase-encoding genes (cel3A and cel4) from the wood-degrading basidiomycete Polyporus arcularius.

In the current study, the genomic and cDNA clones encoding the endoglucanase (cel4) of Polyporus arcularius were sequenced and characterized. The amino acid sequence of Cel4 indicated that it is a glycosyl hydrolase family 5 protein. The expressions of the previously cloned endoglucanase cel3A and cel4 were induced by Avicel (microcrystalline cellulose) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a low level of transcription of both genes regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel3A and cel4 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

Evolutionary changes in defensive specialized metabolism in the genus Hordeum.

Plants have developed defensive specialized metabolites over the course of evolution. In the genus Hordeum, which includes the important cereal crop barley, specialized metabolites such as hordatines, benzoxazinones, and gramine have been identified. Hordeum species are classified into four clades, H, Xu, Xa, and I. The presence or absence of defensive specialized metabolites was analyzed in representative Hordeum species that included all of the four clades. In the H clade, Hordeum vulgare accumulated hordatines but not benzoxazinones, whereas H. bulbosum accumulated neither compound. Some accessions in the H clade accumulated gramine. Species in the clades I and Xa accumulated benzoxazinones without hordatines. In H. murinum, a Xu clade species, neither hordatines nor benzoxazinones were detected. Two hitherto undescribed compounds were found to commonly accumulate in H. bulbosum in the H clade and H. murinum in the Xu clade. On the basis of spectroscopic analyses, they were identified as dehydrodimers of feruloylagmatine and were designated murinamides A and B. Radical coupling reactions with feruloylagmatine as a substrate by peroxidase afforded murinamides A and B. These compounds showed antifungal activities against Bipolaris sorokiniana and Fusarium asiaticum, indicating their defensive roles. Because hordatines are also dehydrodimers of hydroxycinnamic acid amides (HCAAs) of agmatine, both the H and Xu clade species are considered to accumulate the same class of compounds. Thus, when the H/Xu clades split from the I/Xa clades during evolution, the defensive metabolites shifted from benzoxazinones to dehydrodimers of agmatine HCAAs plus gramine in the H/Xu clades.

Anomeric O-acylation of Kdo using alkyl and aryl isocyanates.

To develop a convenient method for the preparation of an alpha-Kdo derivative carrying a functional spacer at the reducing end, we examined anomeric O-acylation using Kdo and halogenated alkyl/aryl isocyanates as nucleophile and electrophiles, respectively. Reaction of a Kdo derivative with 2-chloroethyl isocyanate in the presence of DMAP gave an alpha-spiro product (82%) and an alpha-Kdo derivative of a dimeric isocyanate adduct (10%). Similar reaction with 4-(chloromethyl)phenyl isocyanate gave only the corresponding alpha-spiro product (81%). The NMR data show that the pyranose rings of both the alkyl and aryl spiro products adopt the 5C2 conformation. Thus, we accomplished alpha-selective anomeric O-acylation by coupling the Kdo derivative with alkyl and aryl isocyanates.