Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat.

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.

Measurements of C6-C8 hydrocarbons at a UK rural site during January 1999. Site evaluation and correlations between species.

Ambient concentrations of C6-C8 aromatic hydrocarbons and n-heptane, determined by gas chromatography with flame ionisation detection, are presented from a winter campaign during January 1999 at a rural site near Leeds. Absolute concentrations are significantly lower than those obtained from the only designated UK rural site (Harwell) in the automated UK hydrocarbon network. Both absolute and relative concentrations of hydrocarbons measured at the site have been interpreted in terms of the arriving back-trajectories. The site is subject to two main airflows during the winter months; relatively polluted air from the southwest and much cleaner air from the northwest. Ratios of hydrocarbon concentrations show evidence of significant chemical processing consistent with chemical removal by OH. Uncertainties in the ages of the trajectories prevent a reliable estimation of the average OH concentration over the trajectory. The dependence of the variance of the hydrocarbon concentrations with their lifetime with respect to removal by OH does not show the expected behaviour.

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions.

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.

Proton/hydrogen transfer affects the S-state-dependent microsecond phases of P680+ reduction during water splitting.

To investigate a possible coupling between P680+ reduction and hydrogen transfer, we studied the effects of H2O/D2O exchange on the P680+ reduction kinetics in the nano- and microsecond domains. We concentrated on studying the period-4 oscillatory (i.e., S-state-related) part of the reduction kinetics, by analyzing the differences between the P680+ reduction curves, rather than the full kinetics. Earlier observations that P680+ reduction kinetics have microsecond components were confirmed: the longest observable lifetime whose amplitude showed period-4 oscillations was 30 microseconds. We found that solvent isotope exchange left the nanosecond phases of the P680+ reduction unaltered. However, a significant effect on the oscillatory microsecond components was observed. We propose that, at least in the S0/S1 and S3/S0 transitions, hydrogen (proton) transfer provides an additional decrease in the free energy of the YZ+P680 state with respect to the YZP680+ state. This implies that relaxation of the state YZ+P680 is required for complete reduction of P680+ and for efficient water splitting. The kinetics of the P680+ reduction suggest that it is intraprotein proton/hydrogen rearrangement/transfer, rather than proton release to the bulk, which is occurring on the 1-30 microseconds time scale.

Metabolism and disposition of the antifolate LY231514 in mice and dogs.

The metabolism and disposition of LY231514 was studied in mice and dogs. LY231514 is a novel pyrrotopyrimidine-based multi-target antifolate (MTA) showing broad in vivo antitumor activity in mouse models and is currently in phase II human clinical trials. Doses (iv) of the compound showed high plasma levels, resulting in AUC values of 30-33 micrograms-hr/ml for mice and dogs after 20 and 7.5 mg/kg doses, respectively. The compound was eliminated rapidly. Half-life values for mice and dogs were about 7 and 2 hr, respectively. In vitro plasma binding measured 56% in mice, 46% in dogs, and 81% in humans. Fecal elimination was the major excretion pathway in mice after single iv doses of [14C]LY231514. Urine constituted the major route of excretion in dogs. Parent LY231514 accounted for the majority of urinary radiocarbon in mice (90%) and dogs (68%). Minor metabolites were found in urine, but the amounts were too small to isolate or identify. Based on an earlier observation that LY231514 photodegraded to produce reaction products having similar retention times as these minor urinary isolates, a photo-oxidation system was developed which in fact produced these metabolites. Subsequently, these photolytically-produced materials were used as standards to identify two novel in vivo metabolites formed by oxidation of the pyrrolo-pyrimidine ring system of LY231514. The oxidative transformations are similar to those observed for tryptophan and other indoles in that the pyrrole ring is oxidized to give an amide; further oxidation cleaves this ring, one ring carbon is lost, and a ketone is formed.

Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes.

The receptor for macrophage colony stimulating factor (CSF-1), the c-fms gene product, is a key determinant in the differentiation of monocytic phagocytes. Dissection of the human and mouse c-fms proximal promoters revealed opposing roles for nuclear protooncogenes in the transcriptional regulation of this gene. On the one hand, c-ets-1, c-ets-2, and the macrophage-specific factor PU.1, but not the ets-factor PEA3, trans-activated the c-fms proximal promoter. On the other hand c-myb repressed proximal promoter activity in macrophages and blocked the action of c-ets-1 and c-ets-2. Basal c-fms promoter activity was almost undetectable in the M1 leukaemia line, which expressed high levels of c-myb, but was activated as cells differentiated in response to leukemia inhibitory factor and expressed c-fms mRNA. The repressor function of c-myb depended on the COOH-terminal domain of the protein. We propose that ets-factors are necessary for the tissue-restricted expression of c-fms and that c-myb acts to ensure correct temporal expression of c-fms during myeloid differentiation.

Comparison of the expression and function of the transcription factor PU.1 (Spi-1 proto-oncogene) between murine macrophages and B lymphocytes.

The expression of mRNA encoding the DNA-binding protein PU.1 (Spi-1) is restricted to B lymphocytes and macrophages. The role of PU.1 in tissue-specific transcriptional regulation in the two cell types was examined by co-transfection of a PU.1 expression plasmid with vectors containing B cell (IgH enhancer) or macrophage-specific (c-fms) transcription control elements. Cotransfection of the PU.1 expression plasmid in MOPC31C B cells trans-repressed the IgH enhancer but trans-activated the c-fms promoter. The latter was insufficient to overcome a block to transcription elongation that determines macrophage-specific c-fms gene expression. In the macrophage line RAW264, PU.1 had no effect on the c-fms promoter, but trans-repressed the activity of a c-fms reporter plasmid containing the transcription attenuator. The effects of PU.1 in both cell types were distinct from those of c-ets-2, a related factor, which trans-activated the c-fms promoter in both B cells and macrophages but also repressed the IgH enhancer. PU.1 was shown to be one of several nuclear proteins that bound a critical cis-acting element of the IgH enhancer, microB, but analysis of nuclear extracts of a wide range of B cell and macrophage lines demonstrated a strong correlation between macrophage phenotype and nuclear PU.1 expression. The data suggest that differences in nuclear PU.1 expression and function between macrophages and B cells may play a role in lineage divergence.

Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice.

Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression.

pA2 values for antagonists of platelet activating factor on aggregation of rabbit platelets.

1. The relative potencies, and equilibrium dissociation constants, for nine antagonists of platelet activating factor (Paf) have been determined on rabbit platelets (in diluted platelet-rich plasma (PRP)) in experiments in which the aggregatory response to Paf was measured. 2. Log concentration-response (% maximum) curves to Paf were obtained in the absence (controls) and presence of different concentrations of each Paf antagonist drug. The antagonists shifted the Paf curves to a higher concentration range and the slopes of the Schild plots, constructed from these data, suggested that the drugs were competitive antagonists of Paf. The slopes of the Schild plots for CV-3988 and SRI 63-119 were greater than 1. 3. The pA2 values (pKB values in parentheses) were: WEB 2086 7.31 (7.63); SRI 63-119 6.95; L-652,731 6.71 (6.73); BN 52021 6.38 (6.47); SRI 63-072 6.36 (6.43); CV-3988 5.87; 48740 RP 4.97 (5.07); ketotifen 4.94 (4.95); thiazinamium 4.73 (4.76). 4. This study provides, for the first time, some functional response data for Paf antagonists (pKB values) which are in an appropriate form for use in classifying putative Paf receptors. The study also provides the comparative potencies of these Paf antagonists in inhibiting Paf-induced platelet aggregation. WEB 2086 was the most potent of the drugs examined.

Microvascular leakage to platelet activating factor in guinea-pig trachea and bronchi.

Conscious guinea-pigs received platelet activating factor (PAF, 0.03-0.25 microgram/kg, i.v.) and colloidal carbon (C, tracer for microvascular leakage). Fifteen min later the animal was killed and C-labelled microvessels (leakage) were detected in the mucosal/submucosal region of tracheal and bronchial sections. PAF was more potent than LTD4 or histamine. The numbers of leaky vessel were dose-related, arterioles and arteries were not affected, leaky vessels were seen from proximal trachea to intrapulmonary bronchi and carbon was not apparent in the alveolar wall. The effect was quick in onset, of short duration, could be repeated, was not produced by lyso-PAF and was unaffected by thrombocytopaenia (produced by an antiserum). Thrombocytopaenia itself did not cause leaky vessels in the airways nor affect histamine responses. Thus, PAF causes an increase in microvascular leakage in the conducting airways, which is not dependent on platelets. The affected vessels are probably postcapillary bronchial venules and PAF may act via an endothelial cell receptor in these microvessels. This leakage effect of PAF in the airways could contribute to features of bronchial asthma.

Structure-activity relationships of dimeric Catharanthus alkaloids. 1. Deacetylvinblastine amide (vindesine) sulfate.

Exploration of the effects of "minor" structural differences on the antitumor activity and toxicity of dimeric Catharanthus alkaloids resulted in the preparation of deacetylvinblastine amide (vindesine, VDS) from either vinblastine (VLB) or deacetylvinblastine. Adequate amounts of vindesine for biological testing were prepared by preferential hydrazinolysis of the C23-ester in the vindoline moiety of VLB, followed by hydrogenolysis of the resulting deacetylvinblastine hydrazide. Vindesine in its activity spectrum against rodent tumor systems resembles vincristine (VCR) rather than its parent VLB, while its neurotoxic potential appears to be less than that of VCR. The experimental models developed to estimate this potential include in vitro measurements of axoplasmic transport effects in the cat sciatic nerve and the estimation of neuromuscular disturbances in chickens and monkeys by vindesine in comparison with VCR. A radioimmunoassay for VLB, VCR, and VDS, developed by means of deacetylvinblastine acid azide, has been used to study the pharmacokinetics of vindesine in man. The clinical investigation of vindesine is in progress. Deacetylvinblastine, in contrast to earlier reports, showed activity against several murine tumor systems.