Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation

To understand the nature and function of bacterial biofilm and the process of its formation, we have performed systematic screening of a complete set of Escherichia coli genes/open reading frames (ORFs) to identify those that affect biofilm development upon over-expression. In contrast to the biofilm of strain AG1 used as a control, some of the genes/ORFs when over-expressed led to the formation of an abnormal biofilm such as thin, mat-like, filamentous or one easily detaching from various surfaces. Disruptants of selected genes were constructed in order to clarify their roles in the different stages of biofilm formation. Our results suggest that diverse metabolic pathways contribute to the development of biofilm.     

In vitro biosynthesis of volicitin in Spodoptera litura

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, originally identified in the regurgitant of Spodoptera exigua, induce damaged corn leaves to release volatile compounds which enable parasitic wasps to locate host caterpillars. Here we demonstrate the in vitro biosynthesis of volicitin for the first time by using gut tissues of Spodoptera litura larvae, as well as N-linolenoyl-L-glutamine. When crop, midgut tissues, peritrophic membrane and gut contents isolated from S. litura were incubated with sodium linolenate and L-[alpha-15N] glutamine, not only 15N-labeled N-linolenoyl-L-glutamine but 15N-labeled volicitin was detected mainly in the midgut incubation by LCMS and LCMSMS analysis. In contrast, there were negligible amounts of the newly biosynthesized compounds in the gut content incubation. Furthermore, the microsomal fraction obtained from the gut tissues clearly showed specific incorporation of glutamine. This substrate selectivity accounts for the exclusive uptake of glutamine by fatty acid amides (FAAs) in the noctuid caterpillars, even though glutamine was not a major component in the regurgitant. Additionally, intensive chemical analyses revealed that more than 20% of glutamine in hemolymph was present as conjugates in gut contents. These results suggest that FAA compounds are actively synthesized by caterpillar tissues and might play important physiological role(s) in glutamine metabolism.     

Analysis of thirteen populations of black cohosh for formononetin.

Black cohosh (Actaea racemosa L. syn. Cimicifuga racemosa (L.) Nutt.), a North American perennial plant, is a promising natural alternative to hormone replacement therapy for treating menopausal symptoms, but the mechanism of action is not understood. The clinical actions of this plant have been attributed to the isoflavonone formononetin since 1985, when its presence was reported in a black cohosh extract. Others have since looked for formononetin, but have not detected it. We looked for formononetin in extracts of black cohosh roots and rhizomes collected in thirteen locations in the eastern United States, including Maryland, New Jersey, New York, North Carolina, Pennsylvania, Virginia, and Tennessee. The rhizome samples were extracted using 80% methanol, and the extracts were partially purified using solid-phase extraction to concentrate any isoflavonoids that might be present. We tested for formononetin in these partially purified samples using thin-layer chromatography and high-performance liquid chromatography with a photodiode array detector and a mass spectrometer. Formononetin was not detected in any of the thirteen plant populations examined. Remifemin, a German product now on the United States market, and CimiPure, a commercially available black cohosh rhizome extract, were also analyzed. We did not detect formononetin, or ononin (formononetin-7-glucoside), in any sample tested by the above-mentioned chemical analyses. Therefore, the clinically observed estrogen-like actions of black cohosh, such as reduction of hot flashes, are likely due to a compound, or combination of compounds, other than formononetin.     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

Alanine-scanning mutation approach for classification of the roles of conserved residues in the activity and substrate affinity of l-carnitine dehydrogenase

l-Carnitine dehydrogenase (CDH) is as an excellent tool for l-carnitine (l-Car) estimation. To date, four CDHs have been identified, that share 45 % homology of their proteins. Here 42 conserved residues of CDH from Xanthomonas translucens (Xt-CDH) were substituted successively with alanine. The resultant mutants were analyzed for catalytic activity. Active mutants were evaluated for their influence on l-Car affinity. Twenty-three mutants with reduced affinity toward l-Car were subjected to detailed kinetic analysis. Analytical data implied that all mutants had increased K _m values. The mutants of R193A, E196A, W199A, R200A, F249A, and F253A that produced the greatest l-Car affinity disruption (K _m > 200-folds of Xt-CDH) clustered near the putative active site. This information can provide a solid basis for the rational design of mutagenic investigation to improve CDHs.     

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.     

Ecological consequences through responses of plant and soil communities to changing winter climate

Community processes are now undergoing substantial reconfiguration because of climate change. Although the effects of climate change on ecosystems are currently a major concern, the issues tightly associated with winter climate change have been underrepresented. Given the importance of winter climate variables and events for determining the spatial distribution of communities and their phenological and physiological responses, and the functional roles of each species, all of which are expected to substantially influence community dis/re-assemble in the future, this review focuses on the ecological responses and consequences of terrestrial communities to changing winter climate. In particular, the effects on processes supported by biological interactions are largely undetermined. In this context, focusing on plant–soil feedback as a major interactive multi-system is worthwhile; these interactions can be disentangled through careful evaluation of the functional roles of organisms involved in the feedback (i.e., plants and soil organisms). The underlying mechanisms are indeed complex because direct (i.e., changes in physical conditions) and indirect pathways (i.e., plant-mediated influences on soil-organisms and vice versa) from winter climate change influence the functionality of future ecosystems. To face these issues, the framework of response–effect-traits deserves research priority since this can define community re-organization as the accumulated responses of individual species, which determines the stability and performance of ecosystem functioning. Thus, research that quantifies functional responses and roles of organisms under a changing climate will continue to be essential for the issues of winter climate change, which may become more serious and significant in the near future.     

Accumulation and decay dynamics of coarse woody debris in a Japanese old-growth subalpine coniferous forest

Far less is known about the coarse woody debris (CWD) stock and decay process in temperate Asia compared with that in boreal and temperate Europe and North America. We estimated coniferous CWD stock (logs and snags), decay rate and process, and fungal species responsible for the decay process in a Japanese subalpine coniferous forest. The CWD mass was 42.4 Mg ha^?1, which was the greatest among the previous data recorded in temperate Asia. The decay rate calculated using the annual input of CWD divided by CWD accumulation was 0.036 year^?1, whereas the decay rate when measured chronosequentially was 0.020–0.023 year^?1. The decay process was divided into two phases characterized by different dominant organic chemical constituents. In the first phase, both acid-unhydrolyzable residue and holocellulose decayed simultaneously, suggestive of the white-rot process. In the second phase, holocellulose was selectively decomposed and AUR accumulated, suggestive of the brown-rot process. Nutrients (N, P, K, Na, Mg, and Ca) were mineralized in the first phase but immobilized in the second phase. The fruiting bodies of 26 taxa of fungi were recorded as occurring on CWD in the study area. Trichaptum abietinum and T. fuscoviolaceum, which dominated in the first phase and are known as white-rot fungi, were assumed to be the main decomposers of lignocellulose in the first phase. Although no known strong wood decomposers dominated the second phase, Laetiporus sulphureus and Oligoporus caesius, known as brown-rot fungi, were expected to participate in the selective decomposition of holocellulose in the second phase.