Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Biomechanics and transgenic wood

Wood, or secondary xylem, is composed mostly of three components-cellulose, hemicelluloses, and lignin. Yet this apparent simplicity is deceiving because the sophisticated arrangement of the components on various structural levels, ranging from intricate molecular architecture to defined cellular arrangements to tissue morphology, makes wood a challenging and interesting subject of biomechanical investigation. Recent advances in genetic transformation, providing easier access to wood of specifically altered composition or structure, have opened new opportunities for research on the intricate relation between material structure and composition and mechanical properties. At the same time, investigations into the mechanical properties have provided new information regarding the structural configuration of wood. The present paper reviews the work conducted in this field and outlines future perspectives and prospects for research.     

Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography-linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS-based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300-500 analytes of polar and nonpolar extracts in approximately 400 experimental samples per week per machine.     

FLUCTUATING ALGAL FOOD POPULATIONS AND THE OCCURRENCE OF LESSER FLAMINGOS (PHOENICONAIAS MINOR) IN THREE KENYAN RIFT VALLEY LAKES1

The last two decades have witnessed increasing episodes of lesser flamingo die‐offs in East Africa. Based on data on phytoplankton composition, biomass, and flamingo population density in three alkaline‐saline lakes of Kenya (Bogoria, Nakuru, and Oloidien) in 2001–2010, this study explored the link between sudden flamingo deaths and fluctuations in algal food quantity and quality. The phytoplankton biomass ranged from 13 to 768 mg · L^?1. Similarly, flamingo numbers varied widely from <1,000 to >500,000 individuals in the study lakes. The dominance of the cyanobacterium Arthrospira fusiformis (Woron.) Komárek et J. W. G. Lund was interrupted at irregular intervals in each lake and replaced partly by populations of different species of the nostocalean Anabaenopsis or by the picoplanktonic chlorophyte Picocystis salinarum Lewin. The populations of Anabaenopsis have the potential of blocking the flamingo food filtration system with their large and slimy colonies; moreover, they are able to produce cyanotoxins. Estimates of flamingo populations suggest that low flamingo numbers coincided with periods of low algal food quantity and/or poor quality. A food deficit can be theorized to have two effects on the flamingos: (i) it weakens them to the point of being susceptible to attacks of infective diseases, such as the ones caused by Mycobacterium avium and Pseudomonas aeruginosa, and (ii) it predisposes them to poisoning by cyanotoxins and pollutants, by reducing their capacity to handle toxic substances. This study therefore concludes that the challenges facing the flamingos are associated with changes in their environment, which affect food and water supply.     

Soluble and surface-bound aminopeptidase in eosinophilic blood cells from Mytilus edulis

This study focused on soluble and surface-bound aminopeptidase (AP) in hemocytes from Mytilus edulis and on the identification of the enzyme-producing blood cells. The cell extract hydrolyzed alanine p-nitroanilide (Ala-pNA) with an optimum between pH 6.4 and 7.0. Following native gradient PAGE of extract, alanyl methoxy-naphthylamide (AMNA) was converted by one band with an estimated molecular weight of 375 kDa; it included at least ten putative AP-isozymes with isoelectric points between pH 4.5 and 5.8. In addition to this soluble form, electron microscopy revealed simultaneous conversion of AMNA on the surface of blood cells. Individual mussels expressed AP-molecules in 23-39% of their hemocytes. These cells were shown to represent eosinophilic granulocytes.     

Rarity,ecological memory,rate of floral change in phytoplankton—and the mystery of the Red Cock

In this article, we attempt to estimate the contemporary phytoplankton species pool of a particular lake, by assessing the rate of floral change over a period of 15 years. Phytoplankton time series data from Lake Stechlin, an oligo-mesotrophic lake in the Baltic Lake District (Germany) were used. Of the 254 algal species recorded during the 15-year of studies with roughly biweekly sampling, 212 species were planktonic. In the individual plankton years, the recorded total number of species changed between 97 and 122, of which the number of dominants (>1% contribution to the annual average of total biomass) was only 10–19. The 15-year cumulative number of species exhibited an almost linear increase after an initial saturation phase. This increase was attributed to two reasons: increase of sample size and immigration of species new to the flora. Based on a probabilistic model developed in this study, we estimated the number of co-existing planktonic species of the lake as some 180, and the rate of floral change as 1–2 species per year. Of these co-existing species, only few maintain the matter–energy processing ecosystem functions in any particular plankton year. Selection of these dominants is probably driven by mesoclimatic cycles, coupled with human-induced forcing, like eutrophication. All others are hiding as an ecological memory, in the sense of the capacity or experiences of past states to influence present or future responses of the community. Data analyses suggest that selection of the ‘memory species’ that show temporary abundance increases over shorter (several years) periods are largely dependent upon the dominants. These results show that interspecific interactions and the particular autecological features of the dominants, together with their effects on the whole ecosystem, act as a major organizing force. Some phytoplankton species, like Planktothrix rubescens, are efficient ecosystem engineers with cascading effects of both a top-down and bottom-up nature. Historical scientific data on Planktothrix blooms in Lake Stechlin suggest cyclic patterns in long-term development of phytoplankton which, as the legend of the Red Cock suggests, dates back much further than scientific archives.     

Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols

1,2-Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a sucrose phosphorylase-catalyzed transfer reaction where sucrose is the substrate and 2-O-α-d-glucopyranosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of sucrose hydrolysis, which occurs in the process as a side reaction of the phosphorylase. In addition to ‘nonproductive’ depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0 M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of sucrose donor. Loss of the glucosyl substrate to ‘glucobiose’ by-products was minimal (<7.5%) under these conditions. The reactivity of other acceptors decreased in the order, 3-methoxy-1,2-propanediol > 1,2-propanediol > 3-allyloxy-1,2-propanediol > 3-(o-methoxyphenoxy)-1,2-propanediol > 3-tert-butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by sucrose phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions.     

Imprecise excision of insertion element IS5 from the fliC gene contributes to flagellar diversity in Escherichia coli

Motile strains of Escherichia coli K12 carrying both a chromosomal fliC-H48 gene and a plasmid encoded fliC-H4 gene express both types of flagellins, which are coassembled into functional flagella. By using flagellar-H48-specific antiserum and a plasmid curing procedure, nonmotile mutants were found that carried an IS5 insertion in the chromosomal fliC-H48 gene. Motile revertants were isolated that showed deletions of the IS5 element together with sections of the fliC-H48 gene resulting in an altered flagellar serotype in these strains. As IS5 elements were found associated with 35 of 53 known H-types in wildtype E. coli strains, this insertion element might play a major role in serotype diversity.