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.

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.

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.

A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

Gymnopilins, a product of a hallucinogenic mushroom, inhibit the nicotinic acetylcholine receptor.

Gymnopilins are substances produced in fruiting bodies of the hallucinogenic mushroom, Gymnopilus junonius. Although, only a few biological effects of gymnopilins on animal tissues have been reported, it is believed that gymnopilins are a key factor of the G. junonius poisoning. In the present study, we found that gymnopilins inhibited ACh-evoked responses in neuronal cell line, PC12 cell, and determine the underlying mechanism. Gymnopilins were purified from wild fruiting bodies of G. junonius collected in Japan. Ca(2+)-imaging revealed that gymnopilins reduced the amplitude of ACh-evoked [Ca(2+)]i rises by about 50% and abolished the ACh responses remaining in the presence of atropine. Gymnopilins greatly reduced the amplitude of [Ca(2+)]i rises evoked by nicotinic ACh receptor agonists, 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP) and nicotine. In the whole-cell voltage clamp recording, gymnopilins inhibited the DMPP-evoked currents, but did not affect the voltage-gated Ca(2+) channel currents. These results indicate that gymnopilins directly act on nicotinic ACh receptors and inhibit their activity. This biological action of gymnopilins may be one of the causes of the G. junonius poisoning.

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.

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.

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2.

The aerobic degradation of 3-N-trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment-MS, and GC-MS showed that trimethylamine was the major metabolite, in addition to beta-alanine betaine (beta-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of beta-AB C-N bonds yielded trimethylamine and alkyl chain.

Transcriptional regulation of two cellobiohydrolase encoding genes (cel1 and cel2) from the wood-degrading basidiomycete Polyporus arcularius.

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription 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 cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

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.