How to quantify plant tolerance to loss of biomass?

In some plant species the whole shoot is occasionally removed, as a result of specialist herbivory, grazing, mowing, or other causes. The plant can adapt to defoliation by allocating more to tolerance and less to growth and defense. Plant tolerance to defoliation (TOL1) is typically measured as the ratio between the average dry weight of a group of damaged plants and a control group of undamaged plants, both measured some time after recovery. We develop a model to clarify what TOL1 actually measures. We advocate keeping regrowth (REG2) and shoot–root ratio, both elements of TOL1, separate in the analysis. Based on a resource trade‐off, exotic Jacobaea vulgaris plants from populations in the USA (no specialist herbivory) are expected to grow faster and be less tolerant than native Dutch populations (with specialist herbivory). Indeed Dutch plants had both a significantly larger fraction biomass in roots and faster regrowth (REG2), while US plants attained the highest weight in the control without defoliation. Using key‐factor analysis, we illustrate how growth rates, regrowth, and shoot–root ratio each contribute to final biomass (plant fitness). Our proposed method gives more insight in the mechanisms that underly plant tolerance against defoliation and how tolerance contributes to fitness.     

New methods to approach immunotherapy of cancer – and strategies of tumours to avoid elimination

Owing to the intense effort of numerous investigators, the number of tumour antigens potentially of use for clinical immunotherapy continues to increase. At the same time, further strategies employed by tumour cells to avoid destruction by the immune system are being uncovered. A combined onslaught to target tumour cells and prevent their “escape” will be required for successful immunotherapy. Progress in this area was the subject of a meeting supported by the European Cancer Research Consortium “EUCAPS”, which was held in London in February 2000. This conference was the second of a series, the first of which was summarised previously in this journal [Pawelec G et al. (1999) Cancer Immunol Immunother 48: 214]. Received: 14 March 2000 / Accepted: 30 March 2000     

How to determine the susceptibility of Meligethes aeneus to neonicotinoids?

Three different bioassays were used to determine the susceptibility to the neonicotinoid Biscaya^® of oilseed rape pollen beetles collected from fields in Bavaria. The one in which the test substance was applied to the inner wall of glass tubes is recommended for future studies on pollen beetles because it is not dependent on the availability of plant material and provides precise information on the amount of insecticide required per unit area.     

Computational study of binding of epothilone A to β-tubulin

Understanding the interactions of epothilones with β-tubulin is crucial for computer aided rational design of macrocyclic drugs based on epothilones and epothilone derivatives. Despite numerous structure-activity relationship investigations we still lack substantial knowledge about the binding mode of epothilones and their derivatives to β-tubulin. In this work, we reevaluated the electron crystallography structure of epothilone A/β-tubulin complex (PDB entry 1TVK) and proposed an alternative binding mode of epothilone A to β-tubulin that explains more experimental facts.     

Use of liposomes as membrane models to evaluate the contribution of drug–membrane interactions to antioxidant properties of etodolac

Abstract

This work stresses the need to combine antioxidant assays and drug–membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO?) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO?) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties – fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

The Possible Role of Polyamines to the Recalcitrance of “Kalamata” Olive Leafy Cuttings to Root

The objective of this study was to investigate the role of endogenous polyamines (PAs) (the sum of free plus soluble conjugate plus insoluble bound) on rooting potential of leafy cuttings of an easy, that is,“Arbequina” and a difficult-to-root olive cultivar, that is, “Kalamata”. Subsamples of cuttings were taken for PAs analysis before planting in the mist system and during the early phases of rhizogenesis (EPR). “Arbequina” exhibited higher initial free and total PA content than “Kalamata”. Spermidine (Spd) was the predominant PA observed in both cultivars. A low content of free putrescine (Put) and Spd was found in both cultivars, whereas spermine (Spm) was occasionally detected. “Arbequina” as well as “Kalamata” exhibited the highest free Put and free Spd in summer and Put was the predominant PA among the free PAs. “Arbequina” exhibited the highest individual and total PAs in spring, followed by those in summer and autumn. In contrast, “Kalamata” had the maximum PAs in summer and the lowest in autumn. Changes in the endogenous content of individual and total PAs during the EPR were also observed. Treatment of “Kalamata” cuttings in autumn with both indole-3-butyric acid (IBA) and Put increased rooting compared to IBA alone. Among the PAs administered, Put was the most effective, whereas Spd and Spm failed to promote rooting. PAs, especially in their free form, seem to be involved in the rooting process of olive cuttings; Put application enhanced the rooting response of the difficult-to-root “Kalamata” olive cultivar.     

Application of Animal Modeling to Biodefense Research

The use of biological agents to attack enemies has substantial historical precedent,and includes documented attempts tocontaminate the wells andreservoirs of enemies withcadavers and animal carcasses;and attemptstoinfect Native Ameri-cans with smallpox via contaminated blankets offered as gifts.Awareness and concern over biological weapons has in-creased greatly as the technological sophistication required to produce related agents has become more global.That theuse of biological agents as a m…     

Binding of doxorubicin to cardiolipin as compared to other anionic phospholipids—An evaluation of electrostatic effects

The binding of doxorubicin to large unilamellar vesicles consisting of cardiolipin or other anionic phospholipids was analyzed in terms of the local drug concentration at the membrane surface, according to the Gouy-Chapman theory. The analysis suggests strong positive binding cooperativity. Part of the drug binds in the uncharged form. The affinity for cardiolipin and other anionic phospholipids is comparable. A binding level of 0.5 doxorubicin per lipid-phosphorus is reached when the local concentration of free doxorubicin monomer-equivalents at the membrane surface is about 0.2–0.7 mM. This contrasts with earlier findings indicating a 300–1000 fold higher affinity for cardiolipin. The present analysis provides an explanation for this apparent discrepancy.     

The binding of inhibitors to α-chymotrypsin

1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Prediction of subcellular locations of proteins: where to proceed?

Since the proposal of the signal hypothesis on protein subcellular sorting, a number of computational analyses have been performed in this field. A typical example is the development of prediction algorithms for the subcellular localization sites of input protein sequences. In this review, we mainly focus on the biological grounds of the prediction methods rather than the algorithmic issues because we believe the former will be more fruitful for future development. Recent advances on the study of protein sorting signals will hopefully be incorporated into future prediction methods. Unfortunately, many of the state-of-the-art methods are published without sufficient objective tests. In fact, a simple test employed in this article shows that the performance of specifically developed predictors is not significantly better than that of a homology search. We suspect that this is a general problem associated with the interpretation of genome sequences, which have evolved through gene duplication and speciation.     

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β(2)-microglobulin (β(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β(2)m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β(2)m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β(2)m fibrils, and that both monomeric β(2)m and fibrillar β(2)m are cytotoxic to these cells. β(2)m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β(2)m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β(2)m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β(2)m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.     

Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB. H. Gürleyen and H. Christiansen contributed equally to this work.     

Application of a predictive approach to estimate exposure to non‐smoking urban sub‐populations to background levels of Benzene in Ontario

An indirect approach was used to estimate exposure to background levels of atmospheric benzene for a selection of Ontario non‐smoking urban sub‐populations. Activity codes obtained from nationally representative time‐budget surveys were allocated to five general microenvironments (home, work (or school), outdoors, commuting, and other indoors) and further combined with inhalation rates corresponding to specific levels of physical activity in order to develop average activity patterns for six sub‐populations believed to be differently exposed to atmospheric benzene in urban areas (children, teenagers, office workers, outdoor workers, transit workers, and seniors). These activity patterns were then combined with representative concentrations of benzene measured in the selected microenvironments in Ontario in order to evaluate exposure. Two metrics were used for this purpose, integrated exposure and potential average daily dose (intake). Potential lifetime average daily doses were also estimated for three composite sub‐groups representing average office, outdoor, and transit workers. A probabilistic approach using a Monte‐Carlo sampling procedure was used in order to estimate possible ranges of exposures experienced by the various sub‐populations. Results obtained suggested that the highest daily integrated exposure (mean: 131 μg‐hrs/m³) was associated with the average transit worker while comparable levels were estimated for the other sub‐populations investigated (mean: 77–86 μg‐hrs/m³). These levels corresponded to 24‐hours time‐weighted average (TWA)‐equivalent concentrations of 5.5 μg/m³ and 3.5 μg/m³, respectively. Statistical distributions of integrated exposures obtained with the probabilistic approach indicated levels as high as 343 μg‐hrs/m³ (97.5th percentile) in the case of the average transit worker, corresponding to TWA‐equivalents in excess of 15 μg/m³. When levels of physical activities and inhalation rates were integrated in the calculation of exposure, the highest potential average daily dose was found to be associated with the average child (mean: 3.1 μg/kg‐day; 97.5th percentile: 6.0 μg/kg‐day) whereas comparable amounts were estimated for teenager and transit workers (mean: 2.1 μg/kg‐day; 97.5th: 4.1 and 6.9 μg/kg‐day, respectively). Indoor microenvironments (home, office/school, other indoors) were identified as the major contributors to total exposure and intake of benzene (≥50%) although their relative importance varied depending on the exposure metric utilized. Potential lifetime average daily doses estimated for transit workers varied from 2.1 (mean) to 5.4 (97.5th) μg/kg‐day. This was slightly higher than those estimated for the average office and outdoor workers (mean: 1.5–1.7 μg/kg‐day). These projections suggest that average non‐smoking children and teenagers are the most exposed sub‐populations among those investigated to background levels of atmospheric benzene as a result of their daily activities. However, these projections must be interpreted with caution in view of uncertainties associated with some of the assumptions adopted, the limited database used to document benzene levels, and as a result of unaccounted sources of emissions which, under certain circumstances, can significantly modify these conclusions.     

Ejection of structural zinc leads to inhibition of γ-butyrobetaine hydroxylase

γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II) dependent oxygenase that catalyses an essential step during carnitine biosynthesis in animals. BBOX is inhibited by ejection of structural zinc by a set of selenium containing analogues. Previous structural analyses indicated that an undisrupted N-terminal zinc binding domain of BBOX is required for catalysis. Ebselen is a relatively potent BBOX inhibitor, an observation which may in part reflect its cardioprotective properties.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Environmental assessment of air to water machines—triangulation to manage scope uncertainty

Purpose

Devices that condense and disinfect water vapour to provide chilled drinking water in office environments, so-called ‘air water generators’ (AWGs), are being marketed as environmentally friendly alternatives to the traditional bottled water cooler. We sought to examine this claim.

Methods

The approach adopted was a preliminary life cycle assessment with performance indicators for the use of energy and water and the emission of greenhouse gases. We compared an AWG with its main market competitor, the traditional bottled water cooler and a simple refrigerator containing a jug of water. Modelling was based on Australian conditions and energy supply. To manage possible scope uncertainty, we borrowed the idea of ‘triangulation’ as defined in the social sciences.

Results and discussion

We found that without a renewable energy supply, the claim of environmental superiority is not supported by quantitative analysis. For each indicator, the AWG's score was typically two to four times higher than the alternatives. Energy consumption was the key issue driving all three indicators.

Conclusions

Considering the principal environmental issues related to these systems, air-to-water machines significantly underperform bottled water coolers. A simple refrigerator has the capacity to perform multiple functions and therefore outperform both the bottled and atmospheric water options once allocation of burdens is considered. These conclusions are supported by all three perspectives examined to manage uncertainty.     

Dynamic Properties of Human α-Synuclein Related to Propensity to Amyloid Fibril Formation

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.     

Cell growth: downstream of Myc - to grow or to cycle?

For many years, Myc function has been linked to the control of cell-cycle progression. Now, increasing evidence shows that Myc also controls cell growth, and that these two processes are regulated independently.