Repetition structure of mammalian nuclear DNA

We have used Fragmentation Sequencing logic to analyse the repetition structure of several large human genomic genes. The method, based on a proposed laboratory scheme for DNA sequencing, detects short sequences which are repeated near, but not necessarily adjacent, to each other (cryptically simple DNA). We find a low frequency of such repeats. There is a slight excess of such repeats in introns over exons, and a slight but significant excess in genomic DNA over random DNA, confirming that cryptically simple sequences are over-represented in the genome. The analysis suggests that Fragmentation Sequencing will be a suitable method for sequencing large mammalian genes.     

Technical Information Sheet No. 13: Simple storage of plant cell cultures in liquid media

H.M. Schumacher and K.A. Malik are with DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 B, 38124 Braunschweig, Germany; F. Van Iren is with the Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, NL 2333 AL Leiden, The Netherlands.     

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors

Background

Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs.

Methods

Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching.

Results

Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC₅₀ values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC₅₀ values are far above the concentrations needed to activate or inhibit the caspases.

Conclusion

The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs.

General significance

Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.     

Kinetic and thermodynamic behaviour of wall-bound peroxidase from wheat leaves infected with stripe rust

We have identified two types of peroxidases (POX), one ionically and one covalently bound to the particulate fraction, in stripe rust-infected and -uninfected wheat (Triticum aestivum L.) leaves. The cell walls contained a high level of POX, of which 73–76% was extractable by 1% NaCl and 24–26% by 5 mM EDTA in infected and non-infected leaves of HD 2329. The NaCl-released POX constituted the predominant fraction. Both NaCl- and EDTA-extracted POX exhibited maximum activity at pH 5.0 and had a K _m (enzyme–substrate affinity measure) value of 1.61–1.70 and 1.64–1.67 mM, respectively, with o-dianisidine as the substrate. The V _max (maximum catalytic rate) in the two extractions ranged between 7.06–7.45 and 6.65–7.82 μmol min⁻¹ g⁻¹ fresh weight. A temperature optimum of 50°C was observed for both the NaCl- and EDTA-released fractions. The two POX fractions showed a differential response to metal ions, suggesting their distinctive nature. Sodium azide inhibited POX activity markedly, which suggested the presence of heme as a prosthetic group. Inhibition of wall-bound POX by iodine and the regeneration of activity by mercaptoethanol suggested the involvement of cysteine in the active site of the enzyme. These two forms showed greater differences in terms of thermodynamic properties, such as the energy of activation (E _a) and enthalpy change (ΔH), while entropy (ΔS) and free energy changes were similar. The results further show that pathogen infection of the leaves of this susceptible wheat cultivar induces an increase in the activity and kinetics of POX, which may be critical in the response of the plant cell to infection.     

Native Plant and Microbial Contributions to a Negative Plant-Plant Interaction

A number of hypotheses have been suggested to explain why invasive exotic plants dramatically increase their abundance upon transport to a new range. The novel weapons hypothesis argues that phytotoxins secreted by roots of an exotic plant are more effective against naïve resident competitors in the range being invaded. The common reed Phragmites australis has a diverse population structure including invasive populations that are noxious weeds in North America. P. australis exudes the common phenolic gallic acid, which restricts the growth of native plants. However, the pathway for free gallic acid production in soils colonized by P. australis requires further elucidation. Here, we show that exotic, invasive P. australis contain elevated levels of polymeric gallotannin relative to native, noninvasive P. australis. We hypothesized that polymeric gallotannin can be attacked by tannase, an enzymatic activity produced by native plant and microbial community members, to release gallic acid in the rhizosphere and exacerbate the noxiousness of P. australis. Native plants and microbes were found to produce high levels of tannase while invasive P. australis produced very little tannase. These results suggest that both invasive and native species participate in signaling events that initiate the execution of allelopathy potentially linking native plant and microbial biochemistry to the invasive traits of an exotic species.Invasive weeds are a major source of agricultural costs due to reduced productivity and the labor expended for weed control. In addition, the extensive use of herbicides to control weed populations has undesirable environmental consequences. Therefore, understanding mechanisms that facilitate exotic plant dispersal and displacement of natives in new ranges is critical to predicting and controlling invasions and may yield insights into the ecological processes that govern homeostasis and perturbation in natural plant communities.Phragmites australis (Cav.) Trin ex. Steud. (common reed) has been present in the United States for at least 10,000 years as a major component of mixed tidal wetland plant communities (Saltonstall, 2002). However, over the past 200 years its distribution and abundance has expanded rapidly and it is now considered one of the most aggressive invasive species in marsh communities in North America. Chloroplast DNA analysis has shown that 13 native North American Phragmites haplotypes exist, while invasive populations possess a single chloroplast DNA haplotype (M) that is also widespread in Europe and Asia (Saltonstall, 2002). These data are supported by nuclear microsatellite DNA analysis (Saltonstall, 2003) and morphological differences that distinguish native, noninvasive from exotic, invasive Phragmites in North America (Saltonstall et al., 2004). When grown under the same conditions, exotic Phragmites has significantly higher aboveground and belowground biomass than native Phragmites (Vasquez et al., 2005; Saltonstall and Stevenson, 2007), and this pattern is typically observed under field conditions as well although exceptions exist (League et al., 2006; Meadows and Saltonstall, 2007). Unfortunately today, only remnant native P. australis populations remain along the Atlantic Coast of North America, indicating the near total displacement of native populations by exotic P. australis.Various hypotheses have been forwarded to explain the rapid invasion of P. australis, of which human activities, stress regimes, and hydrologic disturbances have received the greatest attention (Chambers et al., 1999). Compared to invasion in terrestrial ecosystems, invasiveness in marsh communities is less well documented and it is still not clear how environmental factors relate to the establishment of specific dominant marsh species. Although allelopathy has been superficially suggested as the main displacing mechanism in P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979), there has been minimal success in characterizing the responsible allelochemical. Interestingly, three triterpenoids (β-amacin, taraxerol, and taraxerone) and a flavone (tricin) have been identified from aerial portions of P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979). Regrettably, none of these identified chemicals were tested for possible allelopathic activity.Previously, we showed that a root exudate component of P. australis roots inhibits seedling growth, and that production of the exudates is higher in the invasive P. australis haplotype (Rudrappa et al., 2007). The active fraction of this exudate was found to be composed of gallic acid (3,4,5-trihydroxybenzoic acid). Gallic acid is toxic to a variety of weeds, crop plant species, and the model plant species Arabidopsis (Arabidopsis thaliana; Rudrappa et al., 2007; Rudrappa and Bais, 2008). Our published results also show the persistence of gallic acid in soil extracts from P. australis-invaded fields, which validates our in vitro results and strongly supports the idea that P. australis'' invasive behavior may partly be due to the exudation of gallic acid in the soil/marsh (Rudrappa et al., 2007). Our studies concur with the earlier established reports of phytotoxicity and persistence of gallic acid in soil (Weidenhamer and Romeo, 2004).Biochemically, the transition from simple galloylglucoses to complex gallotannins is marked by addition of further galloyl moieties to the pentagalloylglucose (Niemetz and Gross, 2005). It is now known that free gallic acid is released from complexed gallotannins by simple hydrolysis reactions, wherein a tannase activity breaks gallate ester to form free gallic acid, ellagic acid, and Glc (Mahoney and Molyneux, 2004). Treatment of fungal tannase from Aspergillus flavus results in hydrolysis of pellicle-localized gallotannin to form gallic acid, and ellagic acid as two phenolic components (Mahoney and Molyneux, 2004). As gallic acid is often complexed as gallotannins (Niemetz and Gross, 2005), we speculated that plant- or microbial-derived tannase may facilitate free gallic acid release in salt marsh soils.Aside from allelopathy, invasive plants may deleteriously affect interactions between rhizospheric microbial communities and native plant species (Klironomos, 2002; Wardle et al., 2004; Callaway et al., 2008) to promote their expansion in new ranges. One specific example is the disruption of interactions between native species and their arbuscular mycorhizae, upon which the native species rely for nutrient acquisition (Stinson et al., 2006). Another recent study suggests that the recruitment or establishment of an altered soil microbial community may negatively impact the ability of native species to survive in the same soils (Batten et al., 2008). Evidences suggest that soil biota have several effects on the success of invasive plants and the interactions are based in part on the biochemistry, i.e. novel biochemical weapons (Callaway and Ridenour, 2004). However, to our knowledge, no previous studies have directly tested whether P. australis or any other exotic plant may exploit the biochemical potential of native plant and microbial communities to release a phytotoxin (gallic acid) from a relatively benign precursor (gallotannin) in the rhizosphere. This report presents evidence that links native plant and microbial biochemistry to the invasive traits of an exotic species.     

Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes

An assessment of energetic costs associated with P-glycoprotein (P-gp)-mediated xenobiotic efflux is important in understanding the energy budgets, tradeoffs, and fitness of organisms inhabiting contaminated environments. Here, a functional characterization and determination of the energetic costs associated with doxorubicin (DOX) efflux was examined in isolated hepatocytes of rainbow trout. The accumulation and efflux of DOX were both concentration dependent. The efflux of DOX over a 3 h incubation period resulted in a significant decrease in intracellular ATP concentrations (maximum decrease 25%) compared to control baseline levels, while significant increases in concentrations of ADP (max. 26%), AMP (max. 36%) and inorganic phosphate (max. 11%). were observed. In addition, significant reductions in the adenylate energy charge ([AEC]: max 11%), and phosphorylation potential ([PP]: max. 53%) were shown in cells incubated with DOX compared to control cells. Inhibition of DOX efflux (max. 61%) by the non-competitive P-gp inhibitor tariquidar (XR9576), demonstrated that changes in ATP, ADP, AMP, inorganic phosphate concentrations, AEC and PP in DOX-exposed hepatocytes were mainly due to P-gp activity. Overall, these results indicate that the exposure of trout hepatocytes to DOX increases energetic and metabolic costs that are associated specifically with P-gp efflux activity.     

Proinflammatory cytokines inhibit human placental 11beta-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways

Excessive fetal exposure to glucocorticoids has been implicated in the etiology of adult metabolic and cardiovascular disease. Placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) may protect the fetus from excessive glucocorticoid exposure. Maternal stress may be accompanied by elevated levels of cortisol and increased proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha)]. We hypothesize that proinflammatory cytokines inhibit human placental 11beta-HSD activity. We incubated explant cultures of term human placental villi in the presence or absence of 10 ng/ml IL-1beta, IL-6, or TNF-alpha, with or without agonists or antagonists of intracellular Ca2+ and adenylyl cyclase. Activity for 11beta-HSD2 was estimated using a radioisotope assay, and mRNA was measured using quantitative RT-PCR. All cytokines significantly (P < or = 0.05) reduced 11beta-HSD2 activity (>75% suppression); maximal inhibition occurred within 2 h and was maintained for at least 24 h. The IL-1beta-induced inhibitory activity was attenuated using a Ca2+ channel blocker (nifedipine), an intracellular Ca2+ antagonist [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate], or the adenylyl cyclase stimulant forskolin. Conversely, 11beta-HSD2 activity was diminished in the presence of the Ca2+ ionophore A-23187 or the adenylyl cyclase inhibitor SQ-22536. mRNA levels for 11beta-HSD2 were not changed by any of the treatments. Proinflammatory cytokines inhibit human placental 11beta-HSD2 activity through a mechanism that involves increased intracellular Ca2+ and inhibition of adenylyl cyclase. This could result in excessive fetal exposure to maternal cortisol. This mechanism might mediate part of the increased risk of metabolic and cardiovascular disease in adult offspring.     

Lipid content and fatty acid distribution in tissues from Portuguese dogfish, leafscale gulper shark and black dogfish

The lipid characterization in tissues from the three deep-sea sharks leafscale gulper shark (Centrophorus squamosus), Portuguese dogfish (Centroscymnus coelolepis) and black dogfish (Centrocyllium fabricii) captured at Hatton Bank in the North Atlantic were examined. The objective was to determine the lipid content and the fatty acid composition in different tissues. In addition, the fatty acid composition in tissues and species was compared. The tissues examined were pancreas, heart, kidney, stomach, spleen and liver. The lipid content was high in liver (40–50%) and ranged from 1% to 5% in the other tissues. The dominant fatty acids were C16:0, C18:1 (n-9), C18:1 (n-7) and C22:6 (n-3) in all tissues. All tissues had a high content of unsaturated fatty acids.