J. Christopher Hall

Pesticide metabolism in plants and microorganisms

Abstract Understanding pesticide metabolism in plants and microorganisms is necessary for pesticide development, for safe and efficient use, as well as for developing pesticide bioremediation strategies for contaminated soil and water. Pesticide biotransformation may occur via multistep processes known as metabolism or cometabolism. Cometabolism is the biotransformation of an organic compound that is not used as an energy source or as a constitutive element of the organism. Individual reactions of degradation–detoxification pathways include oxidation, reduction, hydrolysis, and conjugation. Metabolic pathway diversity depends on the chemical structure of the xenobiotic compound, the organism, environmental conditions, metabolic factors, and the regulating expression of these biochemical pathways. Knowledge of these enzymatic processes, especially concepts related to pesticide mechanism of action, resistance, selectivity, tolerance, and environmental fate, has advanced our understanding of pesticide science, and of plant and microbial biochemistry and physiology. There are some fundamental similarities and differences between plant and microbial pesticide metabolism. In this review, directed to researchers in weed science, we present concepts that were discussed at a symposium of the American Chemical Society (ACS) in 1999 and in the subsequent book Pesticide Biotransformation in Plants and Microorganism: Similarities and Divergences, edited by J. C. Hall, R. E. Hoagland, and R. M. Zablotowicz, and published by Oxford University Press, 2001. Nomenclature: American Chemical Society; fenchlorazole-ethyl; glutathione; glutathione-S-transferase; naphthalic anhydride; polyaromatic hydrocarbons; polychlorinated biphenyls; reductive dehalogenation; trichloroethylene.

Applications of single-chain variable fragment antibodies in therapeutics and diagnostics

Antibodies (Abs) are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical applications. This review will show the tremendous versatility and importance of scFv fragments as they provide the basic antigen binding unit for a multitude of engineered Abs for use as human therapeutics and diagnostics.

A flavanone and two phenolic acids from Chrysanthemum morifolium with phytotoxic and insect growth regulating activity.

Leaves of Chrysanthemum morifolium cv. Ramat were extracted sequentially with hexane, ethyl acetate, and methanol. The methanol fraction, when incorporated into artificial diet was found to reduce the growth of cabbage looper (Trichoplusia ni Hubner) larvae at concentrations between 500 and 5000 ppm of diet. Fractionation of the methanol extract on a Sephadex column yielded five fractions, three of which reduced the weight of larvae relative to the control. One fraction was analyzed using high performance liquid chromatography (HPLC) and found to contain three main constituents. These compounds were purified using a combination of gel permeation chromatography on Sephadex LH20 and HPLC, and analyzed by 1H and 13C NMR as well as undergoing chemical and physical analyses. The compounds were identified as: 1, chlorogenic acid (5-O-caffeoylquinic acid); 2, 3,5-O-dicaffeoylquinic acid; and 3, 3′, 4′, 5-trihydroxyflavanone 7-O-glucuronide (eriodictyol 7-O-glucuronide). At concentrations between 100 to 1000 ppm these compounds reduced both growth and photosynthesis of Lemna gibba L. with the order of efficacy being: flavanone > chlorogenic acid > 3,5-O-dicaffeoylquinic acid. Furthermore, when incorporated separately into artificial diet these compounds, at 10 to 1000 ppm, enhanced or reduced growth of the cabbage looper (Trichoplusia ni) and gypsy moth (Lymantria dispar L.).

Evolution of Resistance to Auxinic Herbicides: Historical Perspectives, Mechanisms of Resistance, and Implications for Broadleaf Weed Management in Agronomic Crops

Abstract Auxinic herbicides are widely used for control of broadleaf weeds in cereal crops and turfgrass. These herbicides are structurally similar to the natural plant hormone auxin, and induce several of the same physiological and biochemical responses at low concentrations. After several decades of research to understand the auxin signal transduction pathway, the receptors for auxin binding and resultant biochemical and physiological responses have recently been discovered in plants. However, the precise mode of action for the auxinic herbicides is not completely understood despite their extensive use in agriculture for over six decades. Auxinic herbicide-resistant weed biotypes offer excellent model species for uncovering the mode of action as well as resistance to these compounds. Compared with other herbicide families, the incidence of resistance to auxinic herbicides is relatively low, with only 29 auxinic herbicide-resistant weed species discovered to date. The relatively low incidence of resistance to auxinic herbicides has been attributed to the presence of rare alleles imparting resistance in natural weed populations, the potential for fitness penalties due to mutations conferring resistance in weeds, and the complex mode of action of auxinic herbicides in sensitive dicot plants. This review discusses recent advances in the auxin signal transduction pathway and its relation to auxinic herbicide mode of action. Furthermore, comprehensive information about the genetics and inheritance of auxinic herbicide resistance and case studies examining mechanisms of resistance in auxinic herbicide-resistant broadleaf weed biotypes are provided. Within the context of recent findings pertaining to auxin biology and mechanisms of resistance to auxinic herbicides, agronomic implications of the evolution of resistance to these herbicides are discussed in light of new auxinic herbicide-resistant crops that will be commercialized in the near future. Nomenclature: Auxinic herbicides; dominant trait; evolution of resistance; fitness cost; herbicide-resistant crops; mode of action; mechanism of resistance; plant growth regulator; recessive trait.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.

A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

2,4-Dichlorophenoxyacetic acid residues in semen of Ontario farmers

Although paternal exposures to environmental toxicants probably play a role in adverse pregnancy outcomes, few data are available on the extent of this exposure. One semen and two 24-h urine samples were collected from 97 Ontario farmers who had recently used the phenoxy herbicides 2,4-D (2.4-dichlorophenoxyacetic acid) and/or MCPA ([4-chloro-2-methylphenoxyl acetic acid). Both samples were analyzed for 2,4-D using an immunoassay-based technique. Approximately 50% of the semen samples had detectable levels of 2, 4-D (> or =5.0 pph (ng/mL)). Semen levels of 2.4-D were correlated more closely with the second of the two urine samples. Although several studies have measured 2.4-D in the urine of applicators, this study is the first to attempt to measure 2,4-D levels in semen. As these pesticides can be excreted in the semen, they could be toxic to sperm cells and be transported to the woman and developing embryo/fetus. Further research is needed to understand how pesticide handling practices can affect semen pesticide residues and the relationship between the levels observed and reproductive health.

Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies

A key requirement for successful immunotherapeutic and immunodiagnostic applications is the availability of antibodies with high affinity and specificity. In the past, polyclonal antibodies from hyperimmunized animals or monoclonal antibodies from hybridoma cell lines were used extensively and profitably in medicine and immunotechnology. Antibody-based diagnostics, such as immunoassays, are also widely accepted because of their high sensitivity and ease of use as compared to conventional chromatographic techniques. While immunoassays have been used to monitor organic chemical contaminants such as pesticides, food preservatives, antibiotics in agricultural and food industries, hapten-specific antibodies with the desired affinity and specificity are generally difficult to obtain. With the advent of recombinant DNA technology, antibody genes can be amplified and selected through phage display, cell surface display, or cell-free display systems. A particularly useful feature common to all these display systems is the linking of the phenotype and genotype of antibodies during selection. This allows easy co-selection of the desired antibodies and their encoding genes based on the binding characteristics of the displayed antibodies. The selected antibody DNA can be further manipulated for high-level expression, post-translation modification, and/or affinity and specificity improvement to suit their particular applications. Several hapten-specific antibodies, which were successfully selected and engineered to high specificity and affinity using display technologies, have been found to be amenable to conventional immunoassay development. In this review, we will examine different formats of immunoassays designed for hapten identification and various display technologies available for antibody selection and improvement.

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

Uniconazole-induced changes in abscisic acid, total amino acids, and proline in Phaseolus vulgaris.

Abstract Fourteen-day-old bean (Phaseolus vulgaris) plants were soil treated with 100 ml of either 10 mg liter−1 uniconazole ((E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol) or 3 mg liter−1 abscisic acid (ABA). Over the next 10 days, the concentration of uniconazole, ABA, total amino acids, and proline in the primary leaves and their stomatal resistance were monitored. In plants treated with exogenous ABA, there was a single transient increase in stomatal resistance with the endogeous concentrations of ABA, proline, and total amino acids increasing to a maximum approximately 2 days after treatment. In the uniconazole-treated plants, the accumulation of uniconazole in the primary leaves was not linear but was biphasic over the duration of the experiments. This correlated very closely to changes in the stomatal resistance. The concentrations of ABA, proline, and total amino acids followed a similar pattern with the first and second peaks occurring 3–4 and 8–9 days after treatment, respectively. These results suggest that the accumulation of ABA may be the direct result of the presence of uniconazole in the primary leaf tissue. Furthermore, the accumulation of proline and other amino acids that have been associated with environmental stress resistance is likely mediated through the uniconazole-induced accumulation of ABA. A radioimmunoassay for the rapid and sensitive quantification of ABA is also outlined.

Understanding auxinic herbicide resistance in wild mustard: physiological, biochemical, and molecular genetic approaches

Abstract The incidence of auxinic herbicide resistance in plants has increased worldwide. Auxinic herbicides were the first selective organic herbicides developed and have been used in agriculture for over 50 yr, primarily for the selective control of broadleaf weeds in cereal crops. However, the mode of action of auxinic herbicides and the molecular basis of auxinic herbicide resistance remain unknown, although an auxin-binding protein (ABP) is proposed to be the primary target site. Using auxinic herbicide-resistant (R) and -susceptible (S) biotypes of wild mustard as a model system, we have extensively studied the mode of action of auxinic herbicides and the resistance mechanisms at the physiological, biochemical, and molecular genetic levels. There are no differences in uptake, transport, and metabolism of auxinic herbicides between the R and S biotypes. Based on these results, as well as the studies on the role of auxin-enhanced ethylene biosynthesis and calcium in mediating the auxinic herbicide resistance, we hypothesize that resistance of the R biotype to auxinic herbicides is due to an altered target site, possibly an auxin receptor. We have identified and characterized a small ABP gene family as well as their cDNAs from both R and S of wild mustard. Amino acid changes were found in the ABP of the R biotype. Functional and mutational analyses of these genes are underway to determine the role of ABP in mediating auxinic herbicide resistance. In this review, we focus on the mode of action of auxinic herbicides and the molecular basis of auxinic herbicide resistance in wild mustard. Nomenclature: 2,4-D; dicamba; MCPA; MCPP; picloram; ACC, 1-aminocyclopropane-1-carboxylic acid; MACC, 1-malonylaminocyclopropane-1-carboxylic acid; wild mustard, Brassica kaber (DC.) L.C.Wheeler SINAR.