Cyclopropane-1,1-dicarboxylate is a slow-, tight-binding inhibitor of rice ketol-acid reductoisomerase

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of the branched-chain amino acids. The reaction catalyzed consists of two stages, the first of which is an alkyl migration from one carbon atom to its neighbour. The likely transition state is therefore a cyclopropane derivative, and cyclopropane-1,1-dicarboxylate(CPD) has been reported to inhibit the Escherichia coli enzyme. In addition, this compound causes the accumulation of the substrate of ketol-acid reductoisomerase in plants. Here, we investigate the inhibition of the purified rice enzyme. The cDNA was cloned, and the recombinant protein was expressed in E. coli, purified and characterized kinetically. The purified enzyme is strongly inhibited by cyclopropane-1,1-dicarboxylate, with an inhibition constant of 90 nM. The inhibition is time-dependent and this is due to the low rate constants for formation (2.63 X 10(5) M-1 min(-1)) and dissociation (2.37 x 10(-2) min(-1)) of the enzyme-inhibitor complex. Other cyclopropane derivatives are much weaker inhibitors while dimethylmalonate is moderately effective. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Herbicide contamination and the potential impact to seagrass meadows in Hervey Bay, Queensland, Australia

Low concentrations of herbicides (up to 70 ng 1(-1)), chiefly diuron (up to 50 ng 1 (-1)) were detected in surface waters associated with inter-tidal seagrass meadows of Zostera muelleri in Hervey Bay, south-cast Queensland, Australia. Diuron and atrazine (up to 1. 1 ng g(-1) dry weight of sediment) were detected in the sediments of these seagrass meadows. Concentration of the herbicides diuron, simazine and atrazine increased in surface waters associated with seagrass meadows during moderate river flow events indicating herbicides were washed from the catchment to the marine environment. Maximum herbicide concentration (sum of eight herbicides) in the Mary River during a moderate river flow event was 4260 ng 1(-1). No photosynthetic stress was detected in seagrass in this study during low river flow. However, with moderate river flow events, nearshore seagrasses are at risk of being exposed to concentrations of herbicides that are known to inhibit photosynthesis. (c) 2004 Elsevier Ltd. All rights reserved.

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Phytotoxicity of surface waters of the Thames and Brisbane River Estuaries: A combined chemical analysis and bioassay approach for the comparison of two systems

The Thames Estuary, UK, and the Brisbane River, Australia, are comparable in size and catchment area. Both are representative of the large and growing number of the world's estuaries associated with major cities. Principle differences between the two systems relate to climate and human population pressures. In order to assess the potential phytotoxic impact of herbicide residues in the estuaries, surface waters were analysed with a PAM fluorometry-based bioassay that employs the photosynthetic efficiency (photosystem II quantum yield) of laboratory cultured microalgae, as an endpoint measure of phytotoxicity. In addition, surface waters were chemically analysed for a limited number of herbicides. Diuron atrazine and simazine were detected in both systems at comparable concentrations. In contrast, bioassay results revealed that whilst detected herbicides accounted for the observed phytotoxicity of Brisbane River extracts with great accuracy, they consistently explained only around 50% of the phytotoxicity induced by Thames Estuary extracts. Unaccounted for phytotoxicity in Thames surface waters is indicative of unidentified phytotoxins. The greatest phytotoxic response was measured at Charing Cross, Thames Estuary, and corresponded to a diuron equivalent concentration of 180 ng L-1. The study employs relative potencies (REP) of PSII impacting herbicides and demonstrates that chemical analysis alone is prone to omission of valuable information. Results of the study provide support for the incorporation of bioassays into routine monitoring programs where bioassay data may be used to predict and verify chemical contamination data, alert to unidentified compounds and provide the user with information regarding cumulative toxicity of complex mixtures. (c) 2005 Elsevier B.V. All rights reserved.

Pesticide and herbicide residues in sediments and seagrasses from the Great Barrier Reef world heritage area and Queensland coast

Pesticides and herbicides including organochlorine compounds have had extensive current and past application by Queensland's intensive coastal agriculture industry as web as for a wide range of domestic, public health and agricultural purposes in urban areas, The persistent nature of these types of compounds together with possible continued illegal use of banned organochlorine compounds raises the potential for continued long-term chronic exposure to plants and animals of the Great Barrier Reef. Sediment and seagrass samples were collected from 16 intertidal and 25 subtidal sampling sites between Torres Strait and Townsville, near Mackay and Gladstone, and in Hervey and Moreton Bays in 1997 and 1998 and analysed for pesticide and herbicide residues. Low levels of atrazine (0.1-0.3 mug kg(-1)), diuron (0.2-10.1 mug kg(-1)), lindane (0.08-0.19 mug kg(-1)), dieldrin (0.05-0.37 mug kg(-1)), DDT (0.05-0.26 mug kg(-1)), and DDE (0.05-0.26 mug kg(-1)) were detected in sediments and/or seagrasses. Contaminants were mainly detected in samples collected along the high rainfall, tropical coast between Townsville and Port Douglas and in Moreton Bay. Of the contaminants detected, the herbicide diuron is of most concern as the concentrations detected have some potential to impact local seagrass communities, (C) 2000 Elsevier Science Ltd. All rights reserved.

Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours. © 2004 Cancer Research UK.

Chemical modification of smectite clays

Today, speciality use organoclays are being developed for an increasingly large number of specific applications. Many of these, including use in cosmetics, polishes, greases and paints, require that the material be free from abrasive impurities so that the product retains a smooth `feel'. The traditional `wet' method preparation of organoclays inherently removes abrasives naturally present in the parent mineral clay, but it is time-consuming and expensive. The primary objective of this thesis was to explore the alternative `dry' method (which is both quicker and cheaper but which provides no refining of the parent clay) as a process, and to examine the nature of the organoclays produced, for the production of a wide range of commercially usable organophilic clays in a facile way. Natural Wyoming bentonite contains two quite different types of silicate surface (that of the clay mineral montmorillonite and that of a quartz impurity) that may interact with the cationic surfactant added in the `dry' process production of organoclays. However, it is oil shale, and not the quartz, that is chiefly responsible for the abrasive nature of the material, although air refinement in combination with the controlled milling of the bentonite as a pretreatment may offer a route to its removal. Ion exchange of Wyoming bentonite with a long chain quaternary ammonium salt using the `dry' process affords a partially exchanged, 69-78%, organoclay, with a monolayer formation of ammonium ions in the interlayer. Excess ion pairs are sorbed on the silicate surfaces of both the clay mineral and the quartz impurity phases. Such surface sorption is enhanced by the presence of very finely divided, super paramagnetic, Fe2O3 or Fe(O)(OH) contaminating the surfaces of the major mineral components. The sorbed material is labile to washing, and induces a measurable shielding of the 29Si nuclei in both clay and quartz phases in the MAS NMR experiment, due to an anisotropic magnetic susceptibility effect. XRD data for humidified samples reveal the interlamellar regions to be strongly hydrophobic, with the by-product sodium chloride being expelled to the external surfaces. Many organic cations will exchange onto a clay. The tetracationic cyclophane, and multipurpose receptor, cyclobis(paraquat-p-phenylene) undergoes ion exchange onto Wyoming bentonite to form a pillared clay with a very regular gallery height. The major plane of the cyclophane is normal to the silicate surfaces, thus allowing the cavity to remain available for complexation. A series of group VI substituted o-dimethoxybenzenes were introduced, and shown to participate in host/guest interactions with the cyclophane. Evidence is given which suggests that the binding of the host structure to a clay substrate offers advantages, not only of transportability and usability but of stability, to the charge-transfer complex which may prove useful in a variety of commercial applications. The fundamental relationship between particle size, cation exchange capacity and chemical composition of clays was also examined. For Wyoming bentonite the extent of isomorphous substitution increases with decreasing particle size, causing the CEC to similarly increase, although the isomorphous substitution site: edge site ratio remains invarient throughout the particle size range studied.

Factors affecting the growth of Cladophora in relation to river pollution

Nuisance growths of Cladophora have been associated with eutrophication. A review of the literature, however, reveals a scarcity of relevant experimental growth studies. Sampling experimental streams reveals that the addition of sewage effluent to good quality water alters the flora from that dominated by Potamogetan crispus to one dominated by CLadophora. Spatial and temporal differences in biomass of taxa present are discussed in the context of accompanying physicochemical data. In laboratory batch culture, growth of unialgal C. glomerata was accompanied by elevation of medium pH - considered largely responsible for the poor growth in such culture. However, appropriate experimental conditions and indices of growth were selected and the effects of various herbicides assessed. Diquat and terbutryne were shown to possess algicidal activity towards Cladophora. A closed continuous culture apparatus was developed: growth proceeded through lag, logarithmic and linear phases. Inoculum size and medium flow rate had significant effects on growth, and were standardized. In continuous culture, specific growth rate increased linearly with increased duration of light per day, up to 24 hours, and increased light intensity, up to 6000 lux - the highest intensity tested. Comparison of field and laboratory results suggests that ammonia toxicity is attributable to the undissociated form. In the laboratory, 185 µg/1 undissociated ammoniacal nitrogen reduced specific growth rate to 50% of that at 10 µg/1 undissociated ammcniacal nitrogen. 0.077-1.057 mg/1 NO2-N had no significant effect on growth. 7.2-15.2 mg/1 NO3-N had no significant effect on specific growth rate. Neither was any nitrate/phosphate interaction significant. At 4.9 mg/1 PO4-1, specific growth rate was only 48% of that at 1.9 g/1 P04-P. The critical medium PO4-P concentration was <0.1 mg/i. Specific growth rate was reduced to 50% of that in natural water by 0.036 mgCu/l, 0.070 mgzn/1 and 1.03 mgPb/l. Metal uptake was evaluated.

Mechanism of action of a tumour derived lipid mobilising factor

Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-α2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10μM of antagonist SR59230A and partially attenuated by 25μM PD098059 (indicating β3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10μM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 cells (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10μM SR59230A indicating a β3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

A non-parametric data envelopment analysis approach for improving energy efficiency of grape production

Grape is one of the world's largest fruit crops with approximately 67.5 million tonnes produced each year and energy is an important element in modern grape productions as it heavily depends on fossil and other energy resources. Efficient use of these energies is a necessary step toward reducing environmental hazards, preventing destruction of natural resources and ensuring agricultural sustainability. Hence, identifying excessive use of energy as well as reducing energy resources is the main focus of this paper to optimize energy consumption in grape production.In this study we use a two-stage methodology to find the association of energy efficiency and performance explained by farmers' specific characteristics. In the first stage a non-parametric Data Envelopment Analysis is used to model efficiencies as an explicit function of human labor, machinery, chemicals, FYM (farmyard manure), diesel fuel, electricity and water for irrigation energies. In the second step, farm specific variables such as farmers' age, gender, level of education and agricultural experience are used in a Tobit regression framework to explain how these factors influence efficiency of grape farming.The result of the first stage shows substantial inefficiency between the grape producers in the studied area while the second stage shows that the main difference between efficient and inefficient farmers was in the use of chemicals, diesel fuel and water for irrigation. The use of chemicals such as insecticides, herbicides and fungicides were considerably less than inefficient ones. The results revealed that the more educated farmers are more energy efficient in comparison with their less educated counterparts. © 2013.

Modifying the yield factor based on more efficient use of fertilizer — The environmental impacts of intensive and extensive agricultural practices

The aim of this article is to draw attention to calculations on the environmental effects of agriculture and to the definition of marginal agricultural yield. When calculating the environmental impacts of agricultural activities, the real environmental load generated by agriculture is not revealed properly through ecological footprint indicators, as the type of agricultural farming (thus the nature of the pollution it creates) is not incorporated in the calculation. It is commonly known that extensive farming uses relatively small amounts of labor and capital. It produces a lower yield per unit of land and thus requires more land than intensive farming practices to produce similar yields, so it has a larger crop and grazing footprint. However, intensive farms, to achieve higher yields, apply fertilizers, insecticides, herbicides, etc., and cultivation and harvesting are often mechanized. In this study, the focus is on highlighting the differences in the environmental impacts of extensive and intensive farming practices through a statistical analysis of the factors determining agricultural yield. A marginal function is constructed for the relation between chemical fertilizer use and yield per unit fertilizer input. Furthermore, a proposal is presented for how calculation of the yield factor could possibly be improved. The yield factor used in the calculation of biocapacity is not the marginal yield for a given area, but is calculated from the real and actual yields, and this way biocapacity and the ecological footprint for cropland are equivalent. Calculations for cropland biocapacity do not show the area needed for sustainable production, but rather the actual land area used for agricultural production. The proposal the authors present is a modification of the yield factor and also the changed biocapacity is calculated. The results of statistical analyses reveal the need for a clarification of the methodology for calculating marginal yield, which could clearly contribute to assessing the real environmental impacts of agriculture.

Modifying the yield factor based on more efficient use of fertilizer — The environmental impacts of intensive and extensive agricultural practices

The aim of this article is to draw attention to calculations on the environmental effects of agriculture and to the definition of marginal agricultural yield. When calculating the environmental impacts of agricultural activities, the real environmental load generated by agriculture is not revealed properly through ecological footprint indicators, as the type of agricultural farming (thus the nature of the pollution it creates) is not incorporated in the calculation. It is commonly known that extensive farming uses relatively small amounts of labor and capital. It produces a lower yield per unit of land and thus requires more land than intensive farming practices to produce similar yields, so it has a larger crop and grazing footprint. However, intensive farms, to achieve higher yields, apply fertilizers, insecticides, herbicides, etc., and cultivation and harvesting are often mechanized. In this study, the focus is on highlighting the differences in the environmental impacts of extensive and intensive farming practices through a statistical analysis of the factors determining agricultural yield. A marginal function is constructed for the relation between chemical fertilizer use and yield per unit fertilizer input. Furthermore, a proposal is presented for how calculation of the yield factor could possibly be improved. The yield factor used in the calculation of biocapacity is not the marginal yield for a given area, but is calculated from the real and actual yields, and this way biocapacity and the ecological footprint for cropland are equivalent. Calculations for cropland biocapacity do not show the area needed for sustainable production, but rather the actual land area used for agricultural production. The proposal the authors present is a modification of the yield factor and also the changed biocapacity is calculated. The results of statistical analyses reveal the need for a clarification of the methodology for calculating marginal yield, which could clearly contribute to assessing the real environmental impacts of agriculture.

Biological activities of compounds from Piper species

It is well established that secondary metabolites play an important role in plant chemical defense. In an effort to find natural herbicides research on plant growth regulatory activity of secondary metabolites has received more and more attention recently. The genus Piper has been an important source for useful secondary metabolites.^ Crude extracts from Piper species inhibited gram-positive bacteria and higher plant growth under laboratory conditions. Bioassay-guided isolation and purification lead to the identification of safrole, a phenylpropene, as the responsible agent for the inhibitory activity. Safrole was found to leach from naturally fallen leaves with water. Mechanisms of plant growth inhibition by safrole were investigated. Disassociation of cell membrane from cell walls was determined to be a major cause.^ Phenylpropenes structurally similar to safrole had similar phytogrowth inhibitory activity. It is postulated that phenylpropanoids are an important group of naturally occurring secondary metabolites in plant-plant interactions. ^

The use of hyphenated spectrometric techniques for the environmental forensic assessment of non-traditional pollutants and degradates in the greater Florida Everglades

A comprehensive investigation of sensitive ecosystems in South Florida with the main goal of determining the identity, spatial distribution, and sources of both organic biocides and trace elements in different environmental compartments is reported. This study presents the development and validation of a fractionation and isolation method of twelve polar acidic herbicides commonly applied in the vicinity of the study areas, including e.g. 2,4-D, MCPA, dichlorprop, mecroprop, picloram in surface water. Solid phase extraction (SPE) was used to isolate the analytes from abiotic matrices containing large amounts of dissolved organic material. Atmospheric-pressure ionization (API) with electrospray ionization in negative mode (ESP-) in a Quadrupole Ion Trap mass spectrometer was used to perform the characterization of the herbicides of interest. ^ The application of Laser Ablation-ICP-MS methodology in the analysis of soils and sediments is reported in this study. The analytical performance of the method was evaluated on certified standards and real soil and sediment samples. Residential soils were analyzed to evaluate feasibility of using the powerful technique as a routine and rapid method to monitor potential contaminated sites. Forty eight sediments were also collected from semi pristine areas in South Florida to conduct screening of baseline levels of bioavailable elements in support of risk evaluation. The LA-ICP-MS data were used to perform a statistical evaluation of the elemental composition as a tool for environmental forensics. ^ A LA-ICP-MS protocol was also developed and optimized for the elemental analysis of a wide range of elements in polymeric filters containing atmospheric dust. A quantitative strategy based on internal and external standards allowed for a rapid determination of airborne trace elements in filters containing both contemporary African dust and local dust emissions. These distributions were used to qualitative and quantitative assess differences of composition and to establish provenance and fluxes to protected regional ecosystems such as coral reefs and national parks. ^