Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.

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The benzyl mesylate was employed to construct the tetrasubstituted tetrahydrofuran lignan with avoiding Friedel-Crafts type of reaction. The optically pure 7,8-trans, 7′,8′-trans, 7,7′-cis, and 8,8′-cis-virgatusin stereoisomers were synthesized. The enantiomeric excess was >>99%.

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The effect of the benzylic structure of lignan on antioxidant activity was evaluated. Secoisolariciresinol (1) and 3,4-bis(4-hydroxy-3-methoxybenzyl)tetrahydrofuran (2), which have two secondary benzylic positions without oxygen, showed the highest antioxidant activity. Optically active verrucosin (4) was synthesized for the first time in this experiment.

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A new caffeic acid cinnamyl ester (1) was isolated from the n-hexane-soluble fraction of an MeOH extract of the gum resin of Ferula assa-foetida L. The structure was determined to be (2E)-3,4-dimethoxycinnamyl-3-(3,4 diacetoxyphenyl) acrylate on the basis of spectroscopic data including 1D- and 2D-NMR. Compound 1 showed moderate activity for inhibiting LPS-induced nitric oxide production in murine macrophage RAW264.7 cells, with an IC₅₀ value of 54.9 μm.

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The tyrosinase inhibitory activity of the extracts of 53 parts of 36 plant species cultivated for edible and medicinal use in Okinawa was investigated. The extract of Nandina domestica showed potent activity among these. The inhibitor in the extract was purified by assay-guided fractionation to give a simple phenol glucoside. Although it was a known compound (4-β-D-glucopyranosyloxybenzoic acid), its inhibitory activity toward tyrosinase is revealed for the first time in this work.

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Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G₁ production. It inhibited aflatoxin G₁ production by Aspergillus parasiticus with an IC₅₀ value of 0.15 μM without inhibiting aflatoxin B₁ production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC₅₀ values of 0.24 and 3.5 μM, respectively.

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Ethyl 4-[2-(6-methyl-3-pyridyloxy)hexyloxy]benzoate (1) and ethyl 4-(2-phenoxyhexyloxy)benzoate (2), which induce precocious metamorphosis in larvae of Bombyx mori, a clear sign of juvenile hormone (JH) deficiency, showed JH activity when topically applied to allatectomized 4th instar larvae of B. mori. Compounds 1 and 2 induced precocious metamorphosis with doses at which they were effective as JH agonists.

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The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.

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β-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of β-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. β-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that β-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in β-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that β-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.

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To evaluate the antitumor and cytotoxic activity of methanol extract of Phyllanthus polyphyllus (MPP) in mice and human cancer cell lines, the antitumor activity of MPP was evaluated against an Ehrlich ascites carcinoma (EAC) tumor model. The activity was assessed using survival time, hematological studies, lipid peroxidation (LPO), antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione S-transferase (GST), solid tumor mass, and short-term in vitro cytotoxicity. The cytotoxic activity of MPP was evaluated using human breast cancer (MCF7), colon cancer (HT29), and liver cancer (HepG2) cell lines Oral administration of MPP (200 and 300 mg/kg) increased the survival time and significantly reduced the solid tumor volume in a dose-dependent manner. Hematological parameters, protein, and packed cellular volume (PCV), which were altered by tumor inoculation, were restored. MPP significantly decreased the levels of LPO, GPx, GST, and significantly increased the levels of SOD and CAT. In a cytotoxicity study against human cancer cell lines, MPP was found to have IC₅₀ values of 27, 42 and 38 μg/ml on MCF-7, HT-29, and HepG2 cells respectively. MPP possessed significant antitumor and cytotoxic activity on EAC and human cancer cell lines.

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Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a ‘rain-out’ shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r²=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars.

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We cloned and sequenced a photoreceptor gene (Le.phrA) from the basidiomycete Lentinula edodes. The product of Le.phrA, Le.PHRA (924 aa residues) contained a serine-rich region, an LOV domain and two PAS domains. It was clearly smaller than other fungal LOV domain-containing blue-light photoreceptors such as Coprinopsis cinerea Dst1 (1,175 aa), Neurospora crassa WC-1 (1,167 aa), and Cryptococcus neoformans WC-1 (1,141 aa). The Le.phrA gene was found to be transcribed at all stages of the fruiting-body formation of L. edodes, but it was most abundantly transcribed in the immature fruiting body. Fully-matured fruiting body also contained relatively large amounts of Le.phrA transcript. Although the transcript level was lower, preprimordial aggregated mycelial cells grown under a light environment contained larger amounts of the transcript than those grown under continuous darkness, suggesting a light-enhanced expression of the Le.phrA gene. Hymenophore-depleted pileus contained a markedly higher level of the transcript than the stipe.

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Antimicrobial peptides, Enbocin and Gloverin isoforms from the silkworm Bombyx mori, were analyzed for expression of these peptide genes. Tissue-specific expression of Enbocin and Bmgloverin isoform genes was observed mainly in the fat body upon injection of Escherichia coli. Peptidoglycan and lipopolysaccharide triggered expression of these genes in vivo. On the other hand, lipid A activated Bmgloverin isoform genes but not Enbocin isoform genes. These results illustrate the fact that expression of Enbocin and Bmgloverin isoform genes is inducible by bacteria and that the effects of bacterial cell wall components on the activation of these peptide genes are not necessarily the same. In addition, selective activation of the Enbocin2, Bmgloverin2, and Bmgloverin4 genes by BmRelB rather than BmRelA was observed, providing additional evidence for the occurrence of selective activation of antimicrobial peptide genes by a Rel protein. These results suggest complex regulatory mechanisms in insect antimicrobial peptide genes by bacterial cell wall components.

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To develop a new drug delivery system, antibacterial 50–900 nm nanoparticles of shell and internal organs from scallops collected off Huksan-Island, Korea, were prepared by dry grind technology respectively. The diameters, identities, and conformations of the scallop shell and internal organ particles were determined with a particle-size analyzer and by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and Raman spectroscopy. The antibacterial properties of the nanoparticles from scallop shell were investigated in the absence and the presence of scallop-shell extract. Bacterial growth was reduced with the supernatant of the nanoparticle scallop-shell extract. Also, the nanoparticles from scallop shell were much more effective as a skin softener than was powder. These facts provide us with guidelines for the study of the size-dependent properties of functional materials as well as for further applications to drug delivery systems (DDSs) and cosmetic raw materials.

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cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M_r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.

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Plastid transformation technology has been used for the analysis and improvement of plastid metabolism. To create a transplastomic plant with a complicated and massive metabolic pathway, it is necessary to introduce a large amount of DNA into the plastid. However, to our knowledge, the largest DNA fragment introduced into a plastid genome was only 7 kbp long and consisted of just three genes. Here we report the introduction of foreign DNA of 23–50 kbp into the tobacco plastid genome with a bacterial artificial chromosome (BAC)-based plastid transformation vector. It was confirmed that the introduced DNA was passed on to the next generation. This is the first description of plastid transformation with a large amount of foreign DNA.

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Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an α1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.

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Plant isoamylase-type starch-debranching enzymes (ISAs) hydrolyze α-1,6-linkages in α-1,4/α-1,6-linked polyglucans. Two ISAs, designated PvISA1/2 and PvISA3, were purified from developing seeds of kidney bean by ammonium sulfate fractionation and several column chromatographic procedures. The enzymes displayed different substrate specificities for polyglucans: PvISA1/2 showed broad chain-length specificities, whereas PvISA3 liberated specific chains with a DP of 2 to 4.

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We constructed a yeast stain, TKY12Ga, in which phosphatidylethanolamine (PE) synthesis can be controlled by means of the carbon source in the medium. When PE synthesis was blocked, its growth was inhibited. However, in the presence of exogenous didecanoyl PE (diC10PE), TKY12Ga grew despite an inability to synthesize PE. Our system, which employs TKY12Ga strain and diC10PE, provides a valuable tool to study the transport and metabolism of PE in yeast.

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The 474-bp long terminal repeat (LTR) of the basidiomycete Lentinula edodes retrotransposon was found to contain a different type of transcription termination–poly (A)-addition signal on each strand: TATGT- - -TATG- - -TCT, first reported in yeast genes, and an AATAA (or ACATAA)- - -GT(Py)-rich sequence, frequently found in mammalian genes.

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The physiologically active substances in apple vinegar have not yet been chemically characterized. We studied the biological functions of apple vinegar produced from crushed apples, and found that the constituent neutral medium-sized α-glycan (NMαG) acts as an antitumor agent against experimental mouse tumors. NMαG is a homoglycan composed of glucose having a molecular weight of about 10,000 and a branched structure bearing α (1-4,6) linkages.
In this study, we clarified the origin of NMαG in apple vinegar by examination of its content in alcohol and acetic acid fermentation products sequentially. We found that NMαG appeared in acetic acid fermentation, but not in alcohol fermentation. Furthermore we investigated NMαG origin using acetic acid fermentation from alcohol fortifiied apple without alcohol fermentation and from raw material with varying amounts of pomace. The results indicate that NMαG originated in the apple fruit body and that its production requires both fermentation processes.

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Bisphenol A [2,2-bis(4-hydoxyphenyl)propane; BPA] is an endocrine disrupter widely used in polycarbonate plastics and epoxy resins. We investigated the effects of orally administered BPA on antigen-specific responses of the naïve immune system.
BPA was orally administered to T cell receptor transgenic mice, and the antigen-specific responses of immune cells were investigated. Administered BPA moderately reduced interleukin (IL)-2, 4, and interferon (IFN)-γ secretion and increases in IgA and IgG2a production.
Additionally, it was found that orally administered BPA increased antigen-specific IFN-γ production of T cells and modified whole antigen presenting cells (APCs) to suppress antigen-specific cytokine production from T cells.
These findings suggest that BPA can augment the Th1-type responses of naïve immune systems, though the bioavailability of orally administered BPA was low in our experiments.

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The isotope ratio of monoterpene hydrocarbons was used to characterize the citrus essential oils from different species and hybrids. Citrus cold-pressed peel oils from Vietnam were analyzed for the composition and isotope ratio of monoterpene hydrocarbons by using gas chromatography-mass spectrometry. A profile of citrus essential oils on the basis of their isotope ratio values and levels of monoterpene hydrocarbons was developed for Vietnamese citrus. The molecular isotope ratios were lower than those calculated from natural abundance of ¹³C and ²H. In addition, the isotope ratio of the base peaks (m⁄z 94/93) was significantly different among the citrus essential oils from different species and hybrids. The results would be applicable for the characterization of citrus essential oils from different origins.

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Glyceraldehyde (GLA) was determined in glucose degradation and glycation. GLA was detected as a decahydroacridine-1,8-dione derivative on reversed phase HPLC using cyclohexane-1,3-dione derivatizing reagent. The glucose-derived GLA level was higher than the glycation-derived GLA level, because GLA was converted to intermediates and advanced glycation end products (AGE) in glycation. GLA was also generated from 3-deoxyglucosone and glucosone as intermediates of glucose degradation and glycation. This study suggests that glyceraldehyde is generated by hyperglycemia in diabetes, and that it is also formed in medicines such as peritoneal dialysis solution.

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Tempeh is a traditional Indonesian soybean-fermented food produced by filamentous fungi, Rhizopus sp. and Fusarium sp. We isolated and sequenced the genomic gene and a cDNA clone encoding a novel protease (FP) from Fusarium sp. BLB. The genomic gene was 856 bp in length and contained two introns. An isolated cDNA clone encoded a protein of 250 amino acids. The predicted amino acid sequence of FP showed highest homology, of 76%, with that of trypsin from Fusarium oxysporum. The hydrolysis activity of FP toward synthetic peptide was higher than that of any other protease tested, including Nattokinases. Furthermore, the thrombolytic activity of FP was about 2.1-fold higher than that of Nattokinase when the concentration of plasminogen was 24 units/ml. These results suggest that FP is superior to Nattokinases in dissolving fibrin when absorbed into the blood.

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We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 μg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 μg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 μg Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 μg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.

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Supercritical fluid SF-CO₂ treatment of Rosemarinus officinalis L. fresh leaves under optimum conditions (80 °C at 5,000 psi) yielded 5.3% of extract supercritical fluid extraction (SFE)-80, in which five major active principles were identified by liquid chromatography/mass spectrometry (LC/MS), viz., rosmarinic acid, carnosol, 12-methoxycarnosic acid, carnosic acid, and methyl carnosate. Total phenolic content was 155.8 mg/ gallic acid equivalent (GAE)/g in SFE-80, which showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging of 81.86% at 0.01 mg/ml. When treated in RAW 264.7, apparent dose-dependent NO inhibition occurred at dosages of 1.56 to 6.25 μg/ml, and more drastically at 12.5 and 25 μg/ml. At 0.5 to 5.0 μg/ml, SFE-80 exhibited dose-dependent viability suppression and significant tumor necrosis factor alpha (TNF-α) production in Hep 3B, whereas no effect was found in Chang liver cells. Furthermore, no effect was observed in RAW 264.7 at dosages of 3.13 to 25 μg/ml, indicating that SFE-80 exhibited a noncytotoxic character. Conclusively, rosemary can be considered an herbal anti-inflammatory and anti-tumor agent.

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Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

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The oleyl moiety in vanilloids is important in activating vanilloid receptor 1 (TRPV1), but there was no ingredient of ginger containing the oleyl moiety in the natural form. We synthesized oleylgingerol and oleylshogaol and then evaluated their potential to activate a rat TRPV1 channel. Oleylgingerol is a stronger TRPV1 agonist than natural gingerols, but oleylshogaol is a weaker agonist than natural shogaols. The difference in structure between oleylgingerol and oleylshogaol is only the hydroxy group at carbon-5. This hydroxy group might have an important role in activating a TRPV1 channel.

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In the course of screening for 17α-hydroxylase/C_17,20-lyase inhibitors from food ingredients, the methanol soluble fraction of green tea and black tea, which were expected to be rich in catechin and theaflavin content, showed potent inhibitory activity. (−)-Epigallocathechin gallate and theaflavin 3-O-gallate with a pirogallol moiety significantly inhibited C_17,20-lyase activity on IC₅₀ values of 24.5 μM and 11.5 μM respectively. They had potent cytotoxicity against human prostate cancer LNCaP cells (IC₅₀=28.1 μM and 37.4 μM).

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The hypotensive effects of Vaccinium ashei reade (blueberry) leaves were studied in vitro and in vivo. Blueberry leaf showed a strong inhibitory effect on angiotensin-converting enzyme activity in vitro. Additionally, feeding of blueberry leaf suppressed the development of essential hypertension in spontaneously hypertensive rats in vivo. These results promise the use of blueberry leaf as a source of dietary hypotensive components.

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The aim of this study was to establish a rapid preparation of plasma membrane from adipocytes and muscle cells to detect translocated glucose transporter (GLUT) 4. A plasma membrane fraction was prepared by sequential centrifugation with buffer containing detergents, and its purity was estimated by detecting insulin receptor β-subunit (IRβ). After insulin stimulus, GLUT4 translocation was observed in 3T3-L1 adipocytes and L6 myotubes. It was found that IRβ and GLUT4 levels on the plasma membrane decreased in adipose and muscle with intake of a 29% lard diet for 14 weeks. Hence, this method should be useful for rapid preparation of the plasma membrane fraction.

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Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.

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In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu²⁺, Fe²⁺ and Fe³⁺ ions, but was found to be enhanced by Mn²⁺ and Mg²⁺ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.

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The bacterial compositions of feces were monitored in the first 2 months for 15 infants born in Japan, including eight subjects who developed allergy by the age of 2 years. Primer sets targeting six predominant bacterial groups in the infant intestine, Bacteroidaceae, Enterobacteriaceae, bifidobacteria, enterococci, lactobacilli, and the Clostridium perfringens group, were used for real-time PCR to quantitate each population in the feces. The population of Bacteroidaceae was significantly higher in the allergic group at the ages of 1 month (P=0.03) and 2 months (P=0.05) than in the non-allergic group, while no statistically significant difference was observed for the other bacterial populations.

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