Shoot dieback in clipped young Golden Leyland (Cupressocyparis leylandii) trees - a physiological mechanism?

Shoot dieback is a problem in frequently trimmed Leyland hedges and is increasingly affecting gardeners’ choice of hedge trees, having a negative effect on a conifer nursery industry. Some damage can be attributed to the feeding by aphids, but it is unclear if there are also underlying physiological causes. In this study, we tested the hypothesis that shoot-clipping of conifer trees during adverse growing conditions (i.e. high air temperature and low soil moisture) could be leading to shoot ‘dieback’. Three-year-old Golden Leyland Cypress (x Cupressocyparis leylandii ‘Excalibur Gold’) plants were subjected to either a well-watered or droughted irrigation regime and placed in either a ‘hot’ (average day temperature = 40°C) or a ‘cool’ (average day temperature = 27°C) glasshouse compartment. Half of the plants from each glasshouse were clipped on Day 14 and again on Day 50. Measurements of soil moisture content (SMC), net CO2 assimilation rate (A), stomatal conductance (gs), branchlet xylem water potential (XWP), plant height and foliage colour were made. Within the clipped and unclipped treatments of both glasshouse compartments, plants from the droughted regime had significantly lower values for A, gs and XWP than those from the well-watered regime. However, there was no difference in these parameters between the hot and cool glasshouse compartments. The trends seen for A, gs and XWP of all treatments generally mirrored changes in SMC indicating a direct effect of water supply on these parameters. By the end of the experiment the overall foliage colour of plants from the hot glasshouse was darker than that of plants from the cool glasshouse and the overall foliage colour was also darker following shoot clipping. In general, shoot clipping led to increases in A, gs XWP and SMC. This may be due to the reduction in total leaf area leading to a greater supply of water for the remaining leaves. No shoot ‘dieback’ was observed in any treatment in response to drought stress or shoot-clipping.

Apical leaf necrosis as a defence mechanism against pathogen attack: effects of high nutrient availability on onset and rate of necrosis

An outdoor experiment was conducted to increase understanding of apical leaf necrosis in the presence of pathogen infection. Holcus lanatus seeds and Puccinia coronata spores were collected from two adjacent and otherwise similar habitats with differing long-term N fertilization levels. After inoculation, disease and necrosis dynamics were observed during the plant growing seasons of 2003 and 2006. In both years high nutrient availability resulted in earlier disease onset, a higher pathogen population growth rate, earlier physiological apical leaf necrosis onset and a reduced time between disease onset and apical leaf necrosis onset. Necrosis rate was shown to be independent of nutrient availability. The results showed that in these nutrient-rich habitats H. lanatus plants adopted necrosis mechanisms which wasted more nutrients. There was some indication that these necrosis mechanisms were subject to local selection pressures, but these results were not conclusive. The findings of this study are consistent with apical leaf necrosis being an evolved defence mechanism.

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated With Cu2+ ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu2+ to Cu+ and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu+ oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides. (C) 2007 Elsevier Inc. All rights reserved.

Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid

The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isofomus of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follcles (n= 146) from 2-20 min diameter were dissected from ovaries of similar to 40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quanti, the relative abundance of different FS isoforms. Follicle growth from 2-6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2-10 turn. From 6-2 min, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2-6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2-6 nun before falling 2-fold from 6 to 17-20 mm. These findings imply a marked increase in relative activin 'tone' around the stage at which dominant follicle,;election occurs. When larger follicles (13-20 mm) were subdivided according to E/P ratio, those with high (> 5) E/P ratio had lower (2-fold; P < 0(.)001) levels of inh-A and act-A in comparison to follicles with low (< 5) E/P ratio, but there were no significant diffierences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26 13 and 17% respectively of total FS. During growth from 2-20 mm the proportion of total FS represented by 605, 41 and 37 kDa isoforms increased similar to 2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1(.)6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin 'tone' accompanies bovine follicle growth from 3-6 min, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.

The mechanism of bacterial indigo reduction

The reduction of water-insoluble indigo by the recently isolated moderate thermophile, Clostridium isatidis, has been studied with the aim of developing a sustainable technology for industrial indigo reduction. The ability to reduce indigo was not shared with C. aurantibutyricum, C. celatum and C. papyrosolvens, but C. papyrosolvens could reduce indigo carmine (5,5-indigosulfonic acid), a soluble indigo derivative. The supernatant from cultures of C. isatidis, but not from cultures of the other bacteria tested, decreased indigo particle size to one-tenth diameter. Addition of madder powder, anthraquinone-2,6-disulfonic acid, and humic acid all stimulated indigo reduction by C. isatidis. Redox potentials of cultures of C. isatidis were about 100 mV more negative than those of C. aurantibutyricum, C. celatum and C. papyrosolvens, and reached –600 mV versus the SCE in the presence of indigo, but potentials were not consistently affected by the addition of the quinone compounds, which probably act by modifying the surface of the bacteria or indigo particles. It is concluded that C. isatidis can reduce indigo because (1) it produces an extracellular factor that decreases indigo particle size, and (2) it generates a sufficiently reducing potential.

The structure and mechanism of serine acetyltransferase from Escherichia coli

Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 Angstrom crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal alpha-helical domain and a carboxyl- terminal left-handed beta-helix. We identify His(158) and Asp(143) as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed beta-helical structures.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar anti proliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked?

The endocrine placenta has a dilemma; it shares the foetal circulation and yet it needs to secrete active peptide hormones into the maternal circulation to control her metabolism to meet the demands of the growing foetus. Simultaneously, it needs to allow the endocrine systems of the foetus to develop independently. This article will describe how peptide hormones are processed from inactive intermediates and will propose a hypothesis of how the placenta has revised this process to protect the foetus from the potentially damaging affects of these products.

Global iron-dependent gene regulation in Escherichia coli - A new mechanism for iron homeostasis

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur ( ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron- transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron- rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron- containing proteins is repressed under iron- restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of Fe-55-labeled E. coli proteins revealed a marked decrease in iron- protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron- sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Direct time-resolved study of the gas-phase reactions of germylene with ethyl- and diethylgermane: absolute rate constants, temperature dependences, and mechanism

Time-resolved studies of germylene, GeH2, generated by the 193 nm laser flash photolysis of 3,4-dimethyl-1-germacyclopent-3-ene, have been carried out to obtain rate constants for its bimolecular reactions with ethyl- and diethylgermanes in the gas phase. The reactions were studied over the pressure range 1-100 Torr with SF6 as bath gas and at five temperatures in the range 297-564 K. Only slight pressure dependences were found for GeH2 + EtGeH3 (399, 486, and 564 K). The high pressure rate constants gave the following Arrhenius parameters: for GeH2 + EtGeH3, log A = -10.75 +/- 0.08 and E-a = -6.7 +/- 0.6 kJ mol(-1); for GeH2 + Et2GeH2, log A = -10.68 +/- 0.11 and E-a = -6.95 +/- 0.80 kJ mol(-1). These are consistent with fast, near collision-controlled, association processes at 298 K. RRKM modeling calculations are, for the most part, consistent with the observed pressure dependence of GeH2 + EtGeH3. The ethyl substituent effects have been extracted from these results and are much larger than the analogous methyl substituent effects in the SiH2 + methylsilane reaction series. This is consistent with a mechanistic model for Ge-H insertion in which the intermediate complex has a sizable secondary barrier to rearrangement.

First crystallographic signature of an acyclic peptide nanorod: molecular mechanism of nanorod formation by a self-assembled tetrapeptide

A terminally protected acyclic tetrapeptide Boc-Aib-Val-Aib-beta-Ala-OMe 1 (Aib: alpha-aminoisobutyric acid, beta-Ala: beta-Alanine) self-assembles into a continuous hydrogen-bonded supramolecular helix with an average diameter of 10Angstrom (1nm) starting from a double bend molecular conformation in crystals and further self-assembly of this supramolecular architecture leads to the formation of polydisperse nanorods of diameters 10-40 nm.

Crumpet structures: experimental and modelling studies

Crumpets are made by heating fermented batter on a hot plate at around 230°C. The characteristic structure dominated by vertical pores develops rapidly: structure has developed throughout around 75% of the product height within 30s, which is far faster than might be expected from transient heat conduction through the batter. Cooking is complete within around 3 min. Image analysis based on results from X-ray tomography shows that the voidage fraction is approximately constant and that there is continual coalescence between the larger pores throughout the product although there is also a steady level of small bubbles trapped within the solidified batter. We report here experimental studies which shed light on some of the mechanisms responsible for this structure, together with some models of key phenomena.Three aspects are discussed here: the role of gas (carbon dioxide and nitrogen) nuclei in initiating structure development; convective heat transfer inside the developing pores; and the kinetics of setting the batter into an elastic solid structure. It is shown conclusively that the small bubbles of carbon dioxide resulting from the fermentation stage play a crucial role as nuclei for pore development: without these nuclei, the result is not a porous structure, but rather a solid, elastic, inedible, gelatinized product. These nuclei are also responsible for the tiny bubbles which are set in the final product. The nuclei form the source of the dominant pore structure which is largely driven by the, initially explosive, release of water vapour from the batter together with the desorption of dissolved carbon dioxide. It is argued that the rapid evaporation, transport and condensation of steam within the growing pores provides an important mechanism, as in a heat pipe, for rapid heat transfer, and models for this process are developed and tested. The setting of the continuous batter phase is essential for final product quality: studies using differential scanning calorimetry and on the kinetics of change in the visco-elastic properties of the batter suggest that this process is driven by the kinetics of gelatinization. Unlike many thermally driven food processes the rates of heating are such that gelatinization kinetics cannot be neglected. The implications of these results for modelling and for the development of novel structures are discussed.

An insight into the mechanism of protein separation by colloidal gas aphrons (CGA) generated from ionic surfactants

Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of beta-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein-surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 degrees C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect. (c) 2006 Elsevier B.V. All rights reserved.