Binding Affinities of Oxidized and Reduced Forms of Tetrahalogenated Benzoquinones to the QB Site in Oxygen-Evolving Photosystem II Particles from Synechococcus elongatus

Binding affinities of the Q_B site for four tetrahalogenatedbenzoquinones (THBQs) were investigated by measuring their abilityto serve as electron acceptors or act as inhibitors of oxygenevolution in Synechococcus photosystem II particles. Iodanil,bromanil and chloranil but not fluoranil induced a rapid oxidationof Q_A^– and doubled the area over the fluorescence inductioncurve, indicating dark oxidation of Q₄₀₀. Analyses of thesetwo THBQ-induced reactions and inhibition of the acceleratedQ_A^– oxidation by DCMU yielded binding constants of thequinones comparable to those determined from measurements ofoxygen evolution. Generally, THBQs bound tightly to the Q_B site.However, the binding affinity varied in a wide range with THBQs.The Q_B site bound iodanil with an extremely high affinity butfluoranil relatively weakly. The hydroquinone forms of the THBQsalso bound to the Q_B site and inhibited Q_A^– oxidationby Q_B. The concentrations of the hydroquinones required for50% inhibition of Q_A^– oxidation suggest that the Q_B sitebinds the hydroquinones more weakly than the corresponding quinonesexcept for fluoranil, which binds to the Q_B site more tightlyin its reduced form than in oxidized one. The abilities of THBQsto function as electron acceptors or inhibitors of oxygen evolution,and as oxidants of Q₄₀₀ in the dark, are discussed in relationto the binding affinities of the quinones to the Q_B site. ⁴Present address: Department of Biology, Faculty of Science,Toho University. Miyama 2-2-1, Funabashi, 274 Japan     

Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti-asialo GM1 (AGM1) antiserum or anti-Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti-CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.     

In Planta Localization of Stilbenes within Picea abies Phloem

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).Open in a separate windowFigure 1.Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.     

Cyclic mismatch binding ligand CMBL4 binds to the 5′-T-3′/5′-GG-3′ site by inducing the flipping out of thymine base

A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4 with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4 were stacked on each other in aqueous solutions. The most efficient binding of CMBL4 to DNA was observed for the sequence 5′-T-3′/5′-GG-3′ (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)_n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4 with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4-bound duplex containing the T/GG site showed that the CMBL4-binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4 compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site.     

Effects of disinfectants against norovirus virus‐like particles predict norovirus inactivation

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti‐NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus‐like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration‐dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS‐PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.     

Antipredator response of pea aphids <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis> (Hemiptera: Aphididae): effects of predation risks from an alternative patch on a current patch

To escape from predators, herbivorous prey could leave their current patch and relocate to an alternative patch. However, when other predators are present on the new patch, prey are again exposed to predation risk. Thus, patch leaving might be affected by the other predators. We studied patch leaving of pea aphids Acyrthosiphon pisum Harris (Hemiptera: Aphididae) in response to ladybird larvae Harmonia axyridis Pallas (Coleoptera: Coccinellidae) on broad bean Vicia faba L. shoots that were offered as patches for aphids. We tested whether shoot leaving was affected by the presence of predators on alternative shoots under laboratory conditions. Odors from alternative shoots were evaluated as possible cues used by aphids to assess predation risk on the shoots. We exposed aphids to odors from alternative shoots with conspecifics plus either adult or larval ladybirds or larval green lacewings Mallada desjardinsi Navas (Neuroptera: Chrysopidae). Shoot leaving was reduced only when adult ladybirds were present on the alternative shoots compared with controls (i.e., no predators on the alternative shoots). Odors of both adult ladybirds and of conspecifics being attacked by ladybird larvae were required for reduced leaving. Hence, predation risks on current and alternative patches might affect the antipredator responses of aphids.     

Correction to: Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Applied Microbiology and Biotechnology - The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7.     

Insolubilization of exogenous primer dextran with 1,3-α-d-glucan synthase from Streptococcus mutans

Abstract The water-insolubilization mechanism of exogenous primer dextran with 1,3-α- d -glucan synthase (EC 2.4.1.-) from Streptococcus mutans was studied. The 1,3-α- d -glucan synthase solution, containing sucrose and exogenous primer dextran, was incubated briefly. Water-insoluble glucan was synthesized. At the same time, water-soluble glucan, mainly derived from exogenous primer dextran, decreased. Linkage analysis data of glucan produced revealed that 1,3-α- d -glucoside bonds increased. Exogenous primer dextran was changed by the action of 1,3-α- d -glucan synthase to water-insoluble glucan. The results suggest that in a short-term reaction system of outside primer-insertion type, the 1,6-α- d -glucoside bond forms the main chain of water-insoluble glucan.     

Immunological properties of the primer-independent glucosyltransferase of Streptococcus mutans serotypes d and g

Streptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.     

Differential regulation of phenylalanine ammonia-lyase genes during anthocyanin synthesis and by transfer effect in carrot cell suspension cultures

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. In carrot ( Daucus carota L. cv. Kurodagasun) suspension culture cells, PAL is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-dichlo-rophenoxyacetic acid and is also induced rapidly and transiently by transferring and diluting cells to fresh medium. Analyses of nucleotide sequences derived from PAL cDNAs revealed that the PAL mRNAs induced by transfer were transcribed from different carrot PAL genes than the PAL mRNAs induced during anthocyanin synthesis. Northern blotting using probes derived from 3'non-coding regions for PAL cDNAs confirmed that different PAL genes were induced during anthocyanin synthesis and after transfer. Induction of different PAL genes occurs in response to differences in the induction trigger.