Eudesmanolides from Pluchea dioscoridis

Reinvestigation of the aerial parts of Pluchea dioscoridis afforded, in addition to known compounds, five new eudesmanolides and a new thiophene derivative. The stereochemistry of one of the eudesmanolides isolated previously has been corrected. The structures were elucidated by spectroscopic methods.     

Alterations in G-proteins in congestive heart failure in cardiomyopathic (UM-X7.1) hamsters

In order to explain the attenuated sympathetic support during the development of heart failure, the status of -adrenergic mechanisms in the failing myocardium was assessed by employing cardiomyopathic hamsters (155–170 days old) at moderate degree of congestive heart failure. The norepinephrine turnover rate was increased but the norepinephrine content was decreased in cardiomyopathic hearts. The number and the affinity of receptors in the sarcolemmal preparations were not changed in these hearts at moderate stage of congestive heart failure. While the basal adenylyl cyclase activity was not altered in sarcolemma, the stimulation of enzyme activity by NaF, forskolin, Gpp(NH)p or epinephrine was depressed in hearts from these cardiomyopathic hamsters. Since G-proteins are involved in modifying the adenylyl cyclase activity, the functional and bioactivities as well as contents of both Gs and Gi proteins were determined in the cardiomyopathic heart sarcolemma. The functional stimulation of adenylyl cyclase by cholera toxin, which activates Gs proteins, was markedly depressed whereas that by Pertussis toxin, which inhibits Gi proteins, was markedly augmented in cardiomyopathic hearts. The cholera toxin and pertussis toxin catalyzed ADP-ribosylation was increased by 37 and 126%, respectively; this indicated increased bioactivities of both Gs and Gi proteins in experimental preparations. The immunoblot analysis suggested 74 and 124% increase in Gs and Gi contents in failing hearts, respectively. These results suggest that depressed adenylyl cyclase activation in cardiomyopathic hamsters may not only be due to increased content and bioactivity of Gi proteins but the functional uncoupling of Gs proteins from the adenylyl cyclase enzyme may also be involved at this stage of heart failure.     

Influence of experimental soil disturbances on the diversity of plants in agricultural grasslands

Aims and Methods Disturbance is supposed to play an important role for biodiversity and ecosystem stability as described by the intermediate disturbance hypothesis (IDH), which predicts highest species richness at intermediate levels of disturbances. In this study, we tested the effects of artificial soil disturbances on diversity of annual and perennial vascular plants and bryophytes in a field experiment in 86 agricultural grasslands differing in land use in two regions of Germany. On each grassland, we implemented four treatments: three treatments differing in application time of soil disturbances and one control. One year after experimental disturbance, we recorded vegetation and measured biomass productivity and bare ground. We analysed the disturbance response taking effects of region and land-use-accompanied disturbance regimes into account.Important findings Region and land-use type strongly determined plant species richness. Experimental disturbances had small positive effects on the species richness of annuals, but none on perennials or bryophytes. Bare ground was positively related to species richness of bryophytes. However, exceeding the creation of 12% bare ground further disturbance had a detrimental effect on bryophyte species richness, which corresponds to the IDH. As biomass productivity was unaffected by disturbance our results indicate that the disturbance effect on species richness of annuals was not due to decreased overall productivity, but rather due to short-term lowered inter- and intraspecific competition at the newly created microsites. Generally, our results highlight the importance of soil disturbances for species richness of annual plants and bryophytes in agricultural grasslands. However, most grasslands were disturbed naturally or by land-use practices and our additional experimental soil disturbances only had a small short-term effect. Overall, total plant diversity in grasslands seemed to be more limited by the availability of propagules rather than by suitable microsites for germination. Thus, nature conservation efforts to increase grassland diversity should focus on overcoming propagule limitation, for instance by additional sowing of seeds, while the creation of additional open patches by disturbance might only be appropriate where natural disturbances are scarce.     

Polymer-based Preparation of Soil Inoculants: Applications to Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal fungi is an important group of soil microorganisms which form beneficial symbiotic associations with roots with a wide range of plants thus improving plant growth, nutrition and health. This paper reviews the current status of preparation and formulation of mycorrhizal inoculum applying polymer materials with determined characteristics. The most widely used methods are based on the entrapment of fungal materials in natural polysaccharide gels. The potential of such inoculant preparations is illustrated by various studies which include immobilization of mycorrhized root pieces, vesicles and spores, in some cases co-entrapped with other plant beneficial microorganisms. Suggestions for further research in this field are also discussed.     

Coordination of (aminoalkyloxymethyl)dimethylphosphine oxides with palladium(II). Crystal structure of trans-bis[2-(dimethylphosphinoylmethoxy-1,1-dimethylethylamine)]palladium(II) dichloride

The synthesis, structure and spectroscopic properties of novel palladium(II) chloro complexes with a series of (aminoalkyloxymethyl)dimethylphosphine oxides (AOPO) are reported. The complexes with general formula PdCl₂(N,N′-AOPO₂) were obtained by the reaction of PdCl₂(CH₃CN)₂ with the ligands in dry ethanol. The crystal structure of the trans-bis[2-(dimethylphosphinoylmethoxy-1,1-dimethylethylamine)]palladium(II) dichloride has been determined from single-crystal X-ray diffraction data. The compound crystallizes in monoclinic crystal system with P2₁/n space group. The square-planar coordination sphere of palladium consists of two N atoms from two aminoalkyldimethylphosphine ligands and two Cl atoms in trans-arrangement. The AOPO ligand has monodentate coordination through the NH₂ group. The Pd-N and Pd-Cl distances are 2.0610(14) and 2.3225(4) Å, respectively. The preparation of complexes with a composition PdCl₂(AOPO)₂ in chloroform solution are also reported.     

IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.     

2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H₂O₂)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H₂O₂, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H₂O₂- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.Plants are frequently exposed to different abiotic stresses, including high light (HL), UV irradiation, heat, cold, and drought. A component common to these stresses is the rapid formation of reactive oxygen species (ROS) as the result of metabolic dysbalances. A major ROS produced under moderate light (ML) and, in particular, HL photooxidative stress conditions was shown to be singlet oxygen, ¹O₂, that is produced in illuminated chloroplasts predominantly at the PSII (Triantaphylidès et al., 2008). Most of the singlet oxygen is quenched by carotenoids and tocopherols or reacts with galactolipids in thylakoid membranes, yielding galactolipid hydroperoxides (Zoeller et al., 2012; Farmer and Mueller, 2013). In addition, superoxide radicals, O₂·⁻, are produced predominantly at the PSI and rapidly dismutate to hydrogen peroxide (H₂O₂) either spontaneously or because of being catalyzed by superoxide dismutase. Hence, lipid peroxides and H₂O₂ are produced close to the photosystems and may damage thylakoid proteins. In this context, 2-Cys peroxiredoxin (PRX) enzymes have been implicated in the reductive detoxification of lipid peroxides and H₂O₂ (König et al., 2002).During photosynthesis, light energy absorbed by PSII is used to split water molecules, and the electrons are channeled from PSII through PSI to ferredoxin (Fd). As a result, electrons flow from water to Fd. The main electron sink reaction is the Fd NADP oxidoreductase-catalyzed production of NADPH that functions as an electron donor to reduce carbon dioxide to sugars. Under HL conditions, excessive excitation energy is dissipated into heat, which was indicated by nonphotochemical quenching of chlorophyll fluorescence. In addition, excessive photosynthetic electrons can be donated from PSI to O₂, yielding O₂·⁻ (Miyake, 2010). This process, the Mehler reaction, creates an alternative electron sink and electron flow. Superoxide anion radicals, O₂·⁻, can be dismutated to O₂ and H₂O₂ by a thylakoid-attached copper/zinc superoxide dismutase (Cu/ZnSOD; Rizhsky et al., 2003). H₂O₂ can then be reduced to water by peroxidases. As a result, O₂ molecules originating from the water-splitting process at PSII are reduced to water by electrons originating from PSI. This process is termed the water-water cycle (WWC) that is thought to protect the photosynthetic apparatus from excessive light and alleviate photoinhibition.In the classical WWC, the Mehler-ascorbate peroxidase (MAP) pathway, ascorbate peroxidases (APXs) have been considered as key enzymes in the reductive detoxification of H₂O₂ in chloroplasts (Kangasjärvi et al., 2008). APXs reduce H₂O₂ to water and oxidize ascorbate to monodehydroascorbate radicals. NADPH functions as an electron donor to regenerate ascorbate by monodehydroascorbate radical reductase. There are two functional APX homologs in plastids: a 33-kD stromal ascorbate peroxidase (sAPX) and a 38-kD thylakoid ascorbate peroxidase (tAPX). The latter tAPX is thought to reside close to the site of H₂O₂ generation at PSI. Surprisingly, knockout-tAPX mutants as well as double mutants lacking both the tAPX and the sAPX exhibited no visible symptoms of stress after long-term (1–14 d) HL (1.000 µmol photons m⁻² s⁻¹) exposure (Giacomelli et al., 2007; Kangasjärvi et al., 2008; Maruta et al., 2010). Moreover, the photosynthetic efficiency of PSII (as judged by the maximum photochemical efficiency of PSII in the dark-adapted state [F_v/F_m]), H₂O₂ production, antioxidant levels (ascorbate, glutathione, and tocopherols), protein oxidation, and anthocyanin accumulation were similar between light-stressed mutant and wild-type plants. Hence, other H₂O₂ detoxification mechanisms can efficiently compensate for the lack of the sAPX and tAPX detoxification system.In addition to APX, glutathione peroxidases and PRXs may reduce H₂O₂ to water. It has been postulated that, in the chloroplast, two highly homologous thylakoid-associated 2-Cys peroxiredoxins (2CPs), 2CPA and 2CPB, can create an alternative ascorbate-independent WWC (Dietz et al., 2006). In support of this concept, HL stress-acclimated tapx sapx double-mutant plants showed increased levels of 2-Cys PRX compared with wild-type plants (Kangasjärvi et al., 2008). Because the two plastidial 2CPA and 2CPB dynamically interact with the stromal side of thylakoid membranes and are capable of reducing peroxides, 2-Cys PRX enzymes may be involved in both H₂O₂ detoxification and reduction of lipid peroxides in thylakoids (König et al., 2002).The reaction mechanism of 2-Cys PRX is highly conserved and involves a Cys residue, which becomes transiently oxidized to sulphenic acid (termed the peroxidatic Cys residue), thereby reducing H₂O₂ to water. The sulphenic acid is subsequently attacked by a second Cys residue, termed resolving Cys residue, yielding an intermolecular disulfide bridge and water (Dietz, 2011).At high peroxide concentrations, the peroxidase function of 2-Cys PRX becomes inactivated through overoxidation, and excess H₂O₂ may function as a redox signal (Puerto-Galán et al., 2013). It has been postulated that 2-Cys PRXs function as a floodgate that allows H₂O₂ signaling only under oxidative stress conditions (Wood et al., 2003; Dietz, 2011; Puerto-Galán et al., 2013). In addition to its function as peroxidase, 2-Cys PRX may also serve as proximity-based thiol oxidases and chaperones (König et al., 2013).The genome of Arabidopsis (Arabidopsis thaliana) contains two 2CP genes. To study 2-Cys PRX function, transgenic plants with reduced 2-Cys PRX levels were generated by antisense suppression (Baier et al., 2000) as well as crossing of transfer DNA (T-DNA) insertion mutants (Pulido et al., 2010). The T-DNA insertion double mutant was shown to contain less than 5% of the wild-type content of 2CPA and no 2CPB. Hence, full knockout lines lacking both 2-Cys PRXs have not yet been established. Under standard growth conditions, 2-Cys PRX double mutants (similar to plastid APX-deficient plants) also did not show a photooxidative stress phenotype that might be because of compensation by alternative H₂O₂ reduction systems (Pulido et al., 2010). Because of the lack of a clear phenotype of the 2-Cys PRX double-knockdown mutant under ML conditions, the physiological functions of 2CPA and 2CPB remain to be elucidated.The main aim of this study was to identify the physiological function of 2CPA and 2CPB under HL stress conditions, when the WWC is of particular importance in protecting the photosynthetic apparatus from photooxidative damage. We investigated mutants completely deficient in 2-Cys PRX (2cpa 2cpb) or tAPX (tapx) and in addition, 2cpa 2cpb tapx triple knockout plants to study the extent of the functional overlap between these enzymes. Results suggest that 2-Cys PRXs are involved in a 2-Cys PRX-dependent WWC that seems to be more important in protecting the photosynthetic apparatus than the tAPX-dependent WWC, the MAP cycle.