Isolation of High-Purity Betanin from Red Beet and Elucidation of Its Antioxidant Activity against Peroxynitrite: An in vitro Study.

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO- reaction, ONOO--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.

Accumulation of squalene in filamentous fungi Trichoderma virens PS1-7 in the presence of butenafine hydrochloride, squalene epoxidase inhibitor: biosynthesis of 13C-enriched squalene.

Squalene is a triterpenoid compound and widely used in various industries such as medicine and cosmetics due to its strong antioxidant and anticancer properties. The purpose of this study is to increase the accumulation of squalene in filamentous fungi using exogeneous butenafine hydrochloride, which is an inhibitor for squalene epoxidase. The detailed settings achieved that the filamentous fungi, Trichoderma virens PS1-7, produced squalene up to 429.93 ± 51.60 mg/L after culturing for 7 days in the medium consisting of potato infusion with glucose at pH 4.0, in the presence of 200 µm butenafine. On the other hand, no squalene accumulation was observed without butenafine. This result indicated that squalene was biosynthesized in the filamentous fungi PS1-7, which can be used as a novel source of squalene. In addition, we successfully obtained highly 13C-enriched squalene by using [U-13C6]-glucose as a carbon source replacing normal glucose.

Sphagnum capillifolium holobiont from a subarctic palsa bog aggravates the potential of nitrous oxide emissions.

Melting permafrost mounds in subarctic palsa mires are thawing under climate warming and have become a substantial source of N2O emissions. However, mechanistic insights into the permafrost thaw-induced N2O emissions in these unique habitats remain elusive. We demonstrated that N2O emission potential in palsa bogs was driven by the bacterial residents of two dominant Sphagnum mosses especially of Sphagnum capillifolium (SC) in the subarctic palsa bog, which responded to endogenous and exogenous Sphagnum factors such as secondary metabolites, nitrogen and carbon sources, temperature, and pH. SC's high N2O emission activity was linked with two classes of distinctive hyperactive N2O emitters, including Pseudomonas sp. and Enterobacteriaceae bacteria, whose hyperactive N2O emitting capability was characterized to be dominantly pH-responsive. As the nosZ gene-harboring emitter, Pseudomonas sp. SC-H2 reached a high level of N2O emissions that increased significantly with increasing pH. For emitters lacking the nosZ gene, an Enterobacteriaceae bacterium SC-L1 was more adaptive to natural acidic conditions, and N2O emissions also increased with pH. Our study revealed previously unknown hyperactive N2O emitters in Sphagnum capillifolium found in melting palsa mound environments, and provided novel insights into SC-associated N2O emissions.

Pinellic Acid Isolated from Quercetin-rich Onions has a Peroxisome Proliferator-Activated Receptor-Alpha/Gamma (PPAR-α/γ) Transactivation Activity.

Quercetin, a flavonol, is a functional compound that is abundant in onions and is known to have antioxidant and anti-inflammatory effects. Quercetin and its glucoside are known to function as peroxisome proliferator-activated receptor (PPAR) ligands and showed high PPAR-α transactivation activity but little PPAR-γ transactivation activity in some reports. In this study, we demonstrated that an aqueous extract of a quercetin-rich onion cultivar increased transactivation activities not only of PPAR-α but also of PPAR-γ. We isolated (9S,12S,13S)-(10E)-9,12,13-trihydroxyoctadec-10-enoic acid (pinellic acid) obtained from the aqueous extract using PPAR-γ transactivation as an index. Furthermore, it was revealed that pinellic acid could transactivate PPAR-α. Our findings are the first report mentioned showing that trihydroxyoctadec-10-enoic acids showed PPAR-α/γ transactivation activities.

Nitrous Oxide Emission in Response to pH from Degrading Palsa Mire Peat Due to Permafrost Thawing.

N2O, a greenhouse gas, is increasingly emitted from degrading permafrost mounds of palsa mires because of the global warming effects on microbial activity. In the present study, we hypothesized that N2O emission could be affected by a change in pH conditions because the collapse of acidic palsa mounds (pH 3.4-4.6) may result in contact with minerogenic ground water (pH 4.8-6.3), thereby increasing the pH. We compared the effects of pH change on N2O emission from cultures inoculated with peat suspensions. Peat samples were collected on a transect from a still intact high part to the collapsing edge of a degrading palsa mound in northwestern Finland, assuming the microbial communities could be different. We adjusted the pH of peat suspensions prepared from a collapsing palsa mound and compared the N2O emission in a pH gradient from 4.5 to 8.5. The collapsing edge had the highest N2O emission from the peat suspensions among all points on the transect under natural acidic conditions (pH 4.5). The N2O emission was reduced with a moderate rise in pH (pH 5.0-6.0) by approximately 85% compared with natural acidic level (pH 4.5). The bacterial communities in acidic cultures differed considerably from those in alkaline cultures. When pH was adjusted to alkaline conditions, N2O-emitting bacteria different from those present in acidic conditions appeared to emit N2O. The bacterial communities could be characterized by changing pH conditions after thawing and collapse of permafrost have contrasting impacts on N2O production that calls for further attention in future studies.

Bidirectional Cell-Cell Communication via Indole and Cyclo(Pro-Tyr) Modulates Interspecies Biofilm Formation.

The extracellular signaling molecule indole plays a pivotal role in biofilm formation by the enteric gammaproteobacterium Escherichia coli; this process is particularly correlated with the extracellular indole concentration. Using the indole-biodegrading betaproteobacterium Burkholderia unamae, we examined the mechanism by which these two bacteria modulate biofilm formation in an indole-dependent manner. We quantified the spatial organization of cocultured microbial communities at the micrometer scale through computational image analysis, ultimately identifying how bidirectional cell-to-cell communication modulated the physical relationships between them. Further analysis allowed us to determine the mechanism by which the B. unamae-derived signaling diketopiperazine cyclo(Pro-Tyr) considerably upregulated indole biosynthesis and enhanced E. coli biofilm formation. We also determined that the presence of unmetabolized indole enhanced the production of cyclo(Pro-Tyr). Thus, bidirectional cell-to-cell communication that occurred via interspecies signaling molecules modulated the formation of a mixed-species biofilm between indole-producing and indole-consuming species. IMPORTANCE Indole is a relatively stable N-heterocyclic aromatic compound that is widely found in nature. To date, the correlations between indole-related bidirectional cell-to-cell communications and interspecies communal organization remain poorly understood. In this study, we used an experimental model, which consisted of indole-producing and indole-degrading bacteria, to evaluate how bidirectional cell-to-cell communication modulated interspecies biofilm formation via intrinsic and environmental cues. We identified a unique spatial patterning of indole-producing and indole-degrading bacteria within mixed-species biofilms. This spatial patterning was an active process mediated by bidirectional physicochemical interactions. Our findings represent an important step in gaining a more thorough understanding of the process of polymicrobial biofilm formation and advance the possibility of using indole-degrading bacteria to address biofilm-related health and industry issues.

Phenazine-1-carboxylic acid (PCA) produced by Paraburkholderia phenazinium CK-PC1 aids postgermination growth of Xyris complanata seedlings with germination induced by Penicillium rolfsii Y-1.

Symbiosis of Penicillium rolfsii Y-1 is essential for the seed germination of Hawaii yellow-eyed grass (Xyris complanata). However, the local soil where the plants grow naturally often suppresses the radicle growth of the seedlings. This radicle growth was drastically restored by coinoculation of Paraburkholderia phenazinium isolate CK-PC1, which is a rhizobacterium of X. complanata. It was found that the isolate CK-PC1 produced phenazine-1-carboxylic acid (PCA, 1) as a major metabolite. The biological effects of PCA (1) were investigated using the seeds of X. complanata and Mung bean (Vigna radiata) and it was uncovered that the symbiosis of the isolate CK-PC1was essential for the postgermination growth of X. complanata and the metabolite PCA (1) might partially contribute to promote the growth of the plants.

Bacterial seed endophyte shapes disease resistance in rice.

Cereal crop production is severely affected by seed-borne bacterial diseases across the world. Locally occurring disease resistance in various crops remains elusive. Here, we have observed that rice plants of the same cultivar can be differentiated into disease-resistant and susceptible phenotypes under the same pathogen pressure. Following the identification of a seed-endophytic bacterium as the resistance-conferring agent, integration of high-throughput data, gene mutagenesis and molecular interaction assays facilitated the discovery of the underlying mode of action. Sphingomonas melonis that is accumulated and transmitted across generations in disease-resistant rice seeds confers resistance to disease-susceptible phenotypes by producing anthranilic acid. Without affecting cell growth, anthranilic acid interferes with the sigma factor RpoS of the seed-borne pathogen Burkholderia plantarii, probably leading to impairment of upstream cascades that are required for virulence factor biosynthesis. The overall findings highlight the hidden role of seed endophytes in the phytopathology paradigm of 'disease triangles', which encompass the plant, pathogens and environmental conditions. These insights are potentially exploitable for modern crop cultivation threatened by globally widespread bacterial diseases.

An Unknown Non-denitrifier Bacterium Isolated from Soil Actively Reduces Nitrous Oxide under High pH Conditions.

A nitrous oxide (N2O)-consuming bacterium isolated from farmland soil actively consumed N2O under high pH conditions. An acetylene inhibition assay did not show the denitrification of N2 to N2O by this bacterium. When N2O was injected as the only nitrogen source, this bacterium did not assimilate N2O. A polymerase chain reaction demonstrated that this bacterium did not have the typical nosZ gene. This bacterium belonged to Chitinophagaceae, but did not belong to known families that include bacteria with the atypical nosZ. This is the first study to show that a non-denitrifier actively reduces N2O, even under high pH conditions.

Seasonal Fluctuations of Aroma Components of Essential Oils from Larix leptolepis.

Conifer resins are used as chemical raw materials for daily necessities. There have been many reports on the aroma components of turpentine oil from rosin, but there has been no reports on fluctuations in the aroma components through spring to late autumn. We speculated that the aroma components in the essential oils of deciduous coniferous larches might fluctuate during maturation of the foliage. In this study, we focused on the aroma components of larch essential oils and we clarified by multivariate analysis how the aroma components fluctuate during leaf maturation. The results of analysis showed that there was a drastic seasonal fluctuation of the chemical components in larch essential oils, especially in senescent leaves in late autumn. Cryptone and linalool were identified as the characteristic aroma components in essential oil from senescent larch leaves.

Isolation and characterization of 1-palmitoyl-2-linoleoyl-sn-glycerol as a hormogonium-inducing factor (HIF) from the coralloid roots of Cycas revoluta (Cycadaceae).

Coralloid roots are specialized tissues of cycads (Cycas revoluta) that are involved in symbioses with nitrogen-fixing Nostoc cyanobacteria. We found that a crude methanolic extract of coralloid roots induced differentiation of the filamentous cell aggregates of Nostoc species into motile hormogonia. Hence, the hormogonium-inducing factor (HIF) was chased using bioassay-based isolation, and the active principle was characterized as a mixture of diacylglycerols (DAGs), mainly composed of 1-palmitoyl-2-linoleoyl-sn-glycerol (1), 1-palmitoyl-2-oleoyl-sn-glycerol (2), 1-stearoyl-2-linolenoyl-sn-glycerol (3), and 1-stearoyl-2-linoleoyl-sn-glycerol (4). Enantioselectively synthesised compound 1 showed a clear HIF activity at 1 nmol (0.6 µg) disc-1 for the filamentous cells, whereas synthesised 2-linoleoyl-3-palmitoyl-sn-glycerol (1') and 1-palmitoyl-2-linoleoyl-rac-glycerol (1/1') were less active than 1. Conversely, synthesised 1-linoleoyl-2-palmitoyl-rac-glycerol (8/8') which is an acyl positional isomer of compound 1 was inactive. In addition, neither 1-monoacylglycerols nor phospholipids structurally related to 1 showed HIF-like activities. As DAGs are protein kinase C (PKC) activators, 12-O-tetradecanoylphorbol-13-acetate (12), urushiol C15:3-Δ10,13,16 (13), and a skin irritant anacardic acid C15:1-Δ8 (14) were also examined for HIF-like activities toward the Nostoc cells. Neither 12 nor 13 showed HIF-like activities, whereas 14 showed an HIF-like activity at 1 nmol/disc. These findings appear to indicate that some DAGs act as hormogonium-inducing signal molecules for filamentous Nostoc cyanobacteria.

Studies on the metabolic regulation of denitrifying bacteria and phytopathogenic microorganisms using chemical agents found in chemical ecology-based phenomena.

An interaction between two different living creatures is often mediated by a chemical substance, along with metabolic or morphological differentiation. Such phenomenon-based investigation of chemical substances sometimes leads to the discovery of a novel signaling substance associated with biological pest control, including pinpoint regulation of fundamental metabolisms. In studies on the metabolic regulation of denitrifying bacteria and phytopathogenic microorganisms, such chemicals linked to the introduction of new ideas and unique approaches for biorational pest controls are described.

pH Stability and Antioxidant Power of CycloDOPA and Its Derivatives.

CycloDOPA (leukodopachrome), a well known metabolite of tyrosine, is a precursor of melanine in mammalian organisms and of the pigment betalain in plants. However, the isolation of cycloDOPA from natural sources has not been widely reported. In the present work, the stabilities of cycloDOPA and cycloDOPA methyl ester at various pH levels were studied. Both compounds were stable under acidic conditions. By contrast, both compounds were unstable when the pH was shifted from neutral to basic to form indole derivatives as major products. Based on the pH stability, cycloDOPA and its derivatives were subjected to the DPPH radical scavenging assay for the first time.

Comparison of Nostocean hormogonium induction and its motility on solid plates between agar and gellan gum at varying gel matrix concentrations.

To establish a sensitive bioassay for Nostocean hormogonium induction, we compared the effectiveness of the morpho-differentiation induction on two gelled plates, agar and gellan gum, for anacardic acid C15:1-Δ8 decyl ester (1) (100 nmol/disc). On BG-110 (nitrogen-free) medium-based 0.6 and 0.8% agar plates, Nostoc sp. strain Yaku-1 isolated from a coralloid root of Cycas revoluta in Yakushima Island showed clear morpho-differentiation from filamentous aggregates into hormogonia, and the induced hormogonia dispersed within 24 h; however, similar hormogonium formation was not observed at agar concentrations of 1.0% or higher. Conversely, hormogonium induction was considerably more pronounced on gellan gum plates than those on agar plates through concentrations ranging from 0.6 to 1.6% even after 12 h of incubation, particularly active on the 0.8-1.0% gellan gum plates. Thus, gellan gum plates can achieve clear results within 12 h and are thus highly useful for primary screening for hormogonium-inducing factors (HIFs).

Ammonium nitrogen content is a dominant predictor of bacterial community composition in an acidic forest soil with exogenous nitrogen enrichment.

Soil pH is a dominant factor affecting bacterial community composition in acidic, neutral, and alkaline soils but not in severely acidic soils (pH<4.5). We conducted a nitrogen (N) addition experiment in the field in severely acidic forest soil to determine the response of the soil bacterial community and identified the dominant factor in determining community composition. Using a high-throughput Illumina HiSeq sequencing platform, we found that high levels of N addition significantly decreased soil bacterial diversity and altered the composition of the soil bacterial community. The addition of nitrogen increased the relative abundance of copiotrophic taxa (Proteobacteria and Actinobacteria phyla) but decreased the relative abundance of oligotrophic taxa (Acidobacteria, Verrucomicrobia, Planctomycetes, and WD272). In particular, the relative abundance of N-cycling-related microbes (e.g., Burkholderia and Rhizomicrobium genera) also increased upon addition of N. Our correlation analysis showed that soil ammonium nitrogen concentration, rather than pH or nitrate nitrogen concentration, was a key environmental parameter determining the composition of the soil bacterial community. However, these bacterial response behaviors were observed only in the dry season and not in the wet season, indicating that high temperature and precipitation in the wet season may alleviate the impact of the addition of N on soil bacterial diversity and community composition. These results suggest that the soil bacterial community shifted to copiotrophic taxa with higher N demands under increased N addition in severely acidic forest soil.

Comparison of Archaeal Communities in Mineral Soils at a Boreal Forest in Finland and a Cold-Temperate Forest in Japan.

Archaeal communities in mineral soils were compared between a boreal forest in Finland and cold-temperate forest in Japan using 16S rRNA gene-targeted high-throughput sequencing. In boreal soils, Thaumarchaeota Group 1.1c archaea predominated and Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were also detected. In temperate soils, Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were dominant members at the subsurface, whereas their dominancy was replaced by Thermoplasmata archaea at the subsoil. An analysis of the ammonia monooxygenase subunit A gene of Archaea also indicated the distribution of Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea in these soils.

Synthesis of Chiral TFA-Protected α-Amino Aryl-Ketone Derivatives with Friedel-Crafts Acylation of α-Amino Acid N-Hydroxysuccinimide Ester.

Chiral N-protected α-amino aryl-ketones are one of the useful precursors used in the synthesis of various biologically active compounds and can be constructed via Friedel-Crafts acylation of N-protected α-amino acids. One of the drawbacks of this reaction is the utilization of toxic, corrosive and moisture-sensitive acylating reagents. In peptide construction via amide bond formation, N-hydroxysuccinimide ester (OSu), which has high storage stability, can react rapidly with amino components and produces fewer side reactions, including racemization. This study reports the first synthesis and utilization of N-trifluoroacetyl (TFA)-protected α-amino acid-OSu as a potential acyl donor for Friedel-Crafts acylation into various arenes. The TFA-protected isoleucine derivative and its diastereomer TFA-protected allo-isoleucine derivative were investigated to check the retention of α-proton chirality in the Friedel-Crafts reaction. Further utilization of OSu in other branched-chain and unbranched-chain amino acids results in an adequate yield of TFA-protected α-amino aryl-ketone without loss of optical purity.

Base-Mediated One-Pot Synthesis of Aliphatic Diazirines for Photoaffinity Labeling.

Aliphatic diazirines have been widely used as prominent photophores for photoaffinity labeling owing to their relatively small size which can reduce the steric effect on the natural interaction between ligands and proteins. Based on our continuous efforts to develop efficient methods for the synthesis of aliphatic diazirines, we present here a comprehensive study about base-mediated one-pot synthesis of aliphatic diazirines. It was found that potassium hydroxide (KOH) can also promote the construction of aliphatic diazirine with good efficiency. Importantly, KOH is cheaper, highly available, and easily handled and stored compared with the previously used base, potassium tert-butoxide (t-BuOK). Gram-scale study showed that it owned great advantages in being used for the large-scale production of aliphatic diazirines. This protocol is highly neat and the desired products can be easily isolated and purified. As the first comprehensive study of the base-mediated one-pot synthesis of aliphatic diazirines, this work provided good insight into the preparation and utilization of diazirine-based photoaffinity labeling probes.

Identification of diacetonamine from soybean curd residue as a sporulation-inducing factor toward Bacillus spp.

Under bioassay-guided investigation, a sporulation-inducing factor (SIF) toward Bacillus spp. was searched for in methanol (MeOH) extracts of soybean curd residues, and diacetonamine (1) was identified as the active compound. SIF was first isolated as a monoacetylated derivative (2, 4.1 mg from 655 g soybean curd residues), and its chemical structure was elucidated by field desorption mass spectrometry, electron ionization mass spectrometry, and nuclear magnetic resonance (NMR) analyses. After 48-h incubation, 40 µM diacetonamine hydrochloride (1b) exhibited sporulation-inducing activity with 35% sporulation frequency toward a Bacillus amyloliquefaciens wild-type strain (AHU 2170), whereas 40 µM diacetone acrylamide (3) showed 99% sporulation induction, which was much higher than that of 1b. Although Bacillus megaterium NBRC 15308 was sporulated by the treatment with 400 µM 1b with 36 and 70% sporulation frequency after 72- and 96-h incubation respectively, 3 at the same concentration showed only 2% sporulation after 72-h incubation. Hence, diacetonamine (1) was characterized as a genuine SIF from soybean curd residues, but it was uncertain whether 1 is a natural product or an artifact. Spores of B. amyloliquefaciens induced by 1b survived after treatment with heating at 95 °C for 10 min, also suggesting that 1 is genuine SIF in soybean curd residue. As sporulation induction is likely linked to activation of antibiotic production in some spore-forming Firmicutes bacteria, compound 1 would be a possible chemical tool to develop an effective fermentation technology in Bacillus species.

Dehydrogenation of the NH-NH Bond Triggered by Potassium tert-Butoxide in Liquid Ammonia.

A novel strategy for the dehydrogenation of the NH-NH bond is disclosed using potassium tert-butoxide (tBuOK) in liquid ammonia (NH3 ) under air at room temperature. Its synthetic value is well demonstrated by the highly efficient synthesis of aromatic azo compounds (up to 100 % yield, 3 min), heterocyclic azo compounds, and dehydrazination of phenylhydrazine. The broad application of this strategy and its benefit to chemical biology is proved by a novel, convenient, one-pot synthesis of aliphatic diazirines, which are important photoreactive agents for photoaffinity labeling.