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2-Azahypoxanthine was isolated from the fairy ring-forming fungus Lepista sordidaas a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordidawere predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordidamycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2-azahypoxanthine-ribonucleotide generated by NOS5.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscoreaspp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC–MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonicabulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonicaCYP90(DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonicabulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonicabulbils rich in diosgenin would be a beneficial natural resource.

RNA-seq analysis of Cupriavidus necatorNH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.Graphical AbstractRNA-seq analysis showed that 3-chlorobenzoate and benzoate induced the expression of genes for aromatic degradation, transport, and/or chemotaxis strongly in Cupriavidus necatorNH9.

Food proteins are a critical source of nutraceutical bioactive peptides that promote health and prevent diseases. Legume seed proteins have been extensively studied to produce peptides with diverse biological activities. Several legume protein hydrolysates and bioactive peptides have been identified that inhibit the activity of α-amylase, α-glucosidase, and dipeptidyl peptidase IV for the purpose of preventing diabetes. Herein, we observed that chickpea peptides (CPP), prepared using the enzymatic digestion of chickpea protein, induced glucose uptake in C2C12 myotubes. Further, liquid chromatography-tandem mass spectrometry and 2-deoxyglucose uptake assay were used to identify a bioactive CPP, defined as CPP737, REGDIIAVPTGVVF, a part of the legumin A-like protein. CPP737 induced AMP-activated protein kinase phosphorylation and glucose transporter type 4 membrane translocation, but not protein kinase B in C2C12 myotubes. Thus, chickpea protein is a potential source of bioactive peptides that can prevent insulin resistance and hyperglycemia through glucose uptake in the skeletal muscle.

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4–9) (its sequence of RRWQWR) with Escherichia colicells and giant unilamellar vesicles (GUVs). LfcinB (4–9) and lissamine rhodamine B red-labeled LfcinB (4–9) (Rh-LfcinB (4–9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. colicells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4–9) to enter E. colicytoplasm, we investigated the interaction of Rh-LfcinB (4–9) with single cells of E. colicontaining calcein using confocal microscopy. We found that Rh-LfcinB (4–9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4–9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4–9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4–9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4–9) can translocate across lipid membrane regions of the plasma membrane of E. colicells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

In this study, we report a more efficient heterologous expression of lectin from Pleurocybella porrigens(PPL) using an Escherichia coli-based expression system. The yield (9.3 mg/L culture broth) of recombinant PPL (rPPL) using this expression system was increased approximately 9-fold compared to our previous study. The rPPL obtained in this study exhibited the same biochemical properties as the native PPL.

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9(CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9(i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9at 6 min in the absence of pore formation, Pentry(6 min), increased with an increase in CF-R9concentration, but the CF-R9concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry(6 min) of CF-R9for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9concentrations. In contrast, a high concentration of CF-R9induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9entered the GUV lumen, so the CF-R9concentration in the lumen was higher. However, CF-R9could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9enters GUVs of various lipid compositions.

A lectin, PPL, was purified from the mushroom Pleurocybella porrigens. The results of SDS–PAGE, gel filtration, and MALDI-TOF-mass of PPL indicated that its molecular mass was 56 kDa, and it was composed of four 14 kDa subunits with no disulfide bonds. In hemagglutination inhibition assay, PPL exhibited the strongest binding specificity toward GalNAc among the mono- and oligo-saccharides tested. Among the glycoproteins, asialo-bovine submaxillary mucin (asialo-BSM) showed the strongest inhibitory effect. In surface plasmon resonance analysis, asialo-BSM, porcine stomach mucin (PSM), and BSM exhibited potent binding affinity. The complete amino acid sequence was determined by amino acid sequencing of intact and of enzyme-digested PPL. The cDNA of PPL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA of the protein consisted of 411 bp, encoding 137 amino acids. This is the first report of isolation of a lectin of the genus Pleurocybella.

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[2H8] Phenylalanine was converted to [2H8] phenylacetaldehyde and [2H8]-2-phenylethanol by two enzymes derived from the flower petals of R. ‘Hoh-Jun,’ these being identified as pyridoxal-5′-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus‐specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.

ABSTRACTA monoclonal antibody (mAb) IR‐2‐1 was raised against a 67‐kDa protein purified from the macronucleus‐specific bacterial symbiont Holospora obtusa of Paramecium caudatum.The mAb was found to react with two bands (31 and 67‐kDa) on gels of H. obtusa.Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsiand P. bursariaas well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR‐2‐1 reacted with a band of 67 kDa in all Parameciumspecies examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa‐free P. caudatumand no label was seen in the nuclear matrix of the macronucleus of air‐dried P. caudatum.These results suggest that the 67‐kDa antigen found in H. obtusawas not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Parameciumspecies is shared by its bacterial symbiont. In contrast to the decorated tubule‐specific mAb, DS‐1, the antigens for IR‐2‐1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium.Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.

β-Glucosidase was partially purified from Rosa‘Hoh-Jun’ petals. The enzyme was highly specific for such β-D-glucopyranosides as 2-phenylethyl β-D-glucopyranoside. The optimal activity was observed at pH 6.0 and 35 °C. The enzymes were composed with two proteins (160 and 155 kDa) by blue native-PAGE, and were classified in a family 1 glucosidase based on LC-MS/MS analyses.

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.