Biomedical subjects

Paul R Ebert

Publications and source records attributed to Paul R Ebert.

11 recordsLinked to original sources

PubMed2006

Comparative toxicity of fumigants and a phosphine synergist using a novel containment chamber for the safe generation of concentrated phosphine gas.

BACKGROUND: With the phasing out of ozone-depleting substances in accordance with the United Nations Montreal Protocol, phosphine remains as the only economically viable fumigant for widespread use. However the development of high-level resistance in several pest insects threatens the future usage of phosphine; yet research into phosphine resistance mechanisms has been limited due to the potential for human poisoning in enclosed laboratory environments. PRINCIPAL FINDINGS: Here we describe a custom-designed chamber for safely containing phosphine gas generated from aluminium phosphide tablets. In an improvement on previous generation systems, this chamber can be completely sealed to control the escape of phosphine. The device has been utilised in a screening program with C. elegans that has identified a phosphine synergist, and quantified the efficacy of a new fumigant against that of phosphine. The phosphine-induced mortality at 20 degrees C has been determined with an LC(50) of 732 ppm. This result was contrasted with the efficacy of a potential new botanical pesticide dimethyl disulphide, which for a 24 hour exposure at 20 degrees C is 600 times more potent than phosphine (LC(50) 1.24 ppm). We also found that co-administration of the glutathione depletor diethyl maleate (DEM) with a sublethal dose of phosphine (70 ppm, <LC(5)), results in a doubling of mortality in C. elegans relative to DEM alone. CONCLUSIONS: The prohibitive danger associated with the generation, containment, and use of phosphine in a laboratory environment has now been substantially reduced by the implementation of our novel gas generation chamber. We have also identified a novel phosphine synergist, the glutathione depletor DEM, suggesting an effective pathway to be targeted in future synergist research; as well as quantifying the efficacy of a potential alternative to phosphine, dimethyl disulphide.

Animals↗

PubMed2006

Disruption of iron homeostasis increases phosphine toxicity in Caenorhabditis elegans.

The aim of this study is to identify the biochemical mechanism of phosphine toxicity and resistance, using Caenorhabditis elegans as a model organism. To date, the precise mode of phosphine action is unclear. In this report, we demonstrate the following dose-dependent actions of phosphine, in vitro: (1) reduction of ferric iron (Fe3+) to ferrous iron (Fe2+), (2) release of iron from horse ferritin, (3) and the peroxidation of lipid as a result of iron release from ferritin. Using in situ hybridization, we show that the ferritin genes of C. elegans, both ferritin-1 and ferritin-2, are expressed along the digestive tract with greatest expression at the proximal and distal ends. Basal expression of the ferritin-2 gene, as determined by quantitative PCR, is approximately 80 times that of ferritin-1. However, transcript levels of ferritin-1 are induced at least 20-fold in response to phosphine, whereas there is no change in the level of ferritin-2. This resembles the reported pattern of ferritin gene regulation by iron, suggesting that phosphine toxicity may be related to an increase in the level of free iron. Indeed, iron overload increases phosphine toxicity in C. elegans at least threefold. Moreover, we demonstrate that suppression of ferritin-2 gene expression by RNAi, significantly increases sensitivity to phosphine. This study identifies similarities between phosphine toxicity and iron overload and demonstrates that phosphine can trigger iron release from storage proteins, increasing lipid peroxidation, leading to cell injury and/or cell death.

Animals↗

PubMed2005

Identification of functional sequences in the pregenomic RNA promoter of the Banana streak virus Cavendish strain (BSV-Cav).

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and "regain"-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-1-like element, the region around -141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease.

Badnavirus↗

PubMed2005

Functional promoter analysis using an approach based on an in vitro evolution strategy.

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The "selective-shotgun" approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning" deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.

Cloning, Molecular↗

PubMed2004

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis.

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Abscisic Acid↗

PubMed2003

Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis.

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

ATP-Binding Cassette Transporters↗

PubMed2003

Analysis of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonist-independent activity.

Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.

Animals↗

PubMed2003

Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen.

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Aging↗

PubMed2003

Invertebrate D2 type dopamine receptor exhibits age-based plasticity of expression in the mushroom bodies of the honeybee brain.

We have isolated a cDNA clone from the honeybee brain encoding a dopamine receptor, AmDop2, which is positively coupled to adenylyl cyclase. The transmembrane domains of this receptor are 88% identical to the orthologous Drosophila D2 dopamine receptor, DmDop2, though phylogenetic analysis and sequence homology both indicate that invertebrate and vertebrate D2 receptors are quite distinct. In situ hybridization to mRNA in whole-mount preparations of honeybee brains reveals gene expression in the mushroom bodies, a primary site of associative learning. Furthermore, two anatomically distinct cell types in the mushroom bodies exhibit differential regulation of AmDop2 expression. In all nonreproductive females (worker caste) and reproductive males (drones) the receptor gene is strongly and constitutively expressed in all mushroom body interneurons with small cell bodies. In contrast, the large cell-bodied interneurons exhibit dramatic plasticity of AmDop2 gene expression. In newly emerged worker bees (cell-cleaning specialists) and newly emerged drones, no AmDop2 transcript is observed in the large interneurons whereas this transcript is abundant in these cells in the oldest worker bees (resource foragers) and older drones. Differentiation of the mushroom body interneurons into two distinct classes (i.e., plastic or nonplastic with respect to AmDop2 gene expression) indicates that this receptor contributes to the differential regulation of distinct neural circuits. Moreover, the plasticity of expression observed in the large cells implicates this receptor in the behavioral maturation of the bee.

Aging↗

PubMed2003

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

Amino Acid Sequence↗

PubMed2002

Genetic linkage analysis of the lesser grain borer Rhyzopertha dominica identifies two loci that confer high-level resistance to the fumigant phosphine.

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.

Animals↗