Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.     

Characterization of inhibitory effects of perfluorooctane sulfonate on human hepatic cytochrome P450 isoenzymes: focusing on CYP2A6

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4'-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited (K(i) value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 (K(i) value of 4 μM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately (K(i) values of 35 to 45 μM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS (K(i) values of 190-300 μM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K(inact) and K(I) values of 0.06 min(-1) and 23 μM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.     

The parasitoid fly Exorista japonica uses visual and olfactory cues to locate herbivore‐infested plants

Some parasitoid flies exploit odors derived from plants as olfactory cues for locating the food plants of host insects, but the role of visual cues associated with plants remains largely unknown. The generalist tachinid Exorista japonica Townsend (Diptera: Tachinidae) is attracted to odors derived from maize plants [Zea mays L. (Poaceae)] infested by the larvae of Mythimna separata (Walker) (Lepidoptera: Noctuidae). In this study, we examined the effects of visual parameters on the olfactory attraction of female flies to host‐infested plants. A paper plant model of one of four colors (blue, green, yellow, or red) was placed in front of a host‐infested plant, which was hidden behind a mesh screen in a wind tunnel. The landing rate of females was significantly higher on the green plant model than on the other three models. When an achromatic plant model of one of four gray scales (white, light gray, dark gray, or black) was tested, the response rate of females was significantly higher towards the white model and decreased as the brightness of models decreased. Few female flies responded to the green plant model without odors of the host‐infested plants. When the four color plant models were placed together in a cage filled with odors of host‐infested plants, females remained significantly longer on the green model than on the other three models. These results showed that E. japonica females preferred the color green when odors of the host‐infested plants were present and suggest that E. japonica uses visual as well as olfactory cues to locate the host habitat.     

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.     

Truncation of N- and C-terminal regions of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> dextranase enhances catalytic activity

Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23–70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.     

Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The K_i values of the enzyme for Cu²⁺ and Cu⁺ ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF–MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Distance decay relationships in foliar fungal endophytes are driven by rare taxa

Foliar fungal endophytes represent a diverse and species‐rich plant microbiome. Their biogeography provides essential clues to their cryptic relationship with hosts and the environment in which they disperse. We present species composition, diversity, and dispersal patterns of endophytic fungi associated with needles of Pinus taeda trees across regional scales in the absence of strong environmental gradients as well as within individual trees. An empirical designation of rare and abundant taxa enlightens us on the structure of endophyte communities. We report multiple distance‐decay patterns consistent with effects of dispersal limitation, largely driven by community changes in rare taxa, those taxonomic units that made up less than 0.31% of reads per sample on average. Distance‐decay rates and community structure also depended on specific classes of fungi and were predominantly influenced by rare members of Dothideomycetes. Communities separated by urban areas also revealed stronger effects of distance on community similarity, confirming that host density and diversity plays an important role in symbiont biogeography, which may ultimately lead to a mosaic of functional diversity as well as rare species diversity across landscapes.