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Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed.

Isolates of the rice blast fungus Magnaporthe grisea that carry the gene encoding Avirulence Conferring Enzyme1 (ACE1) are specifically recognized by rice (Oryza sativa) cultivars carrying the resistance gene Pi33. This recognition enables resistant plants to activate a defense response. ACE1 was isolated by map-based cloning and encodes a putative hybrid between a polyketide synthase and a nonribosomal peptide synthetase, enzymes involved in microbial secondary metabolism. ACE1 is expressed exclusively during fungal penetration of host leaves, the time point at which plant defense reactions are triggered. Ace1 appears to be localized in the cytoplasm of the appressorium. Mutation of the putative catalytic site of the beta-ketoacyl synthase domain of Ace1 abolishes recognition of the fungus by resistant rice. This suggests that Ace1 biosynthetic activity is required for avirulence. Our results are consistent with the hypothesis that the fungal signal recognized by resistant rice plants is the secondary metabolite whose synthesis depends on Ace1.

In higher plants, very-long-chain fatty acids (VLCFAs) are the main constituents of hydrophobic polymers that prevent dessication at the leaf surface and provide stability to pollen grains. Of the 21 genes encoding VLCFA elongases (VLCFAEs) from Arabidopsis thaliana, 17 were expressed heterologously in Saccharomyces cerevisiae. Six VLCFAEs, including three known elongases (FAE1, KCS1, and KCS2) and three previously uncharacterized gene products (encoded by At5g43760, At1g04220, and At1g25450) were found to be enzymatically active with endogenous yeast fatty acid substrates and to some extent with externally supplied unsaturated substrates. The spectrum of VLCFAs accumulated in expressing yeast strains was determined by gas chromatography/mass spectrometry. Marked specificity was found among elongases tested with respect to their elongation products, which encompassed saturated and monounsaturated fatty acids 20-30 carbon atoms in length. The active VLCFAEs revealed highly distinct patterns of differential sensitivity to oxyacetamides, chloroacetanilides, and other compounds tested, whereas yeast endogenous VLCFA production, which involves its unrelated elongase (ELO) in sphingolipid synthesis, was unaffected. Several compounds inhibited more than one VLCFAE, and some inhibited all six active enzymes. These findings pinpoint VLCFAEs as the target of the widely used K(3) class herbicides, which have been in commercial use for 50 years, provide important clues as to why spontaneous resistance to this class is rare, and point to complex patterns of substrate specificity and product spectrum among members of the Arabidopsis VLCFAE family.

An analytical method for the simultaneous quantitation of arseneous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO) in human urine by coupling of high-performance liquid chromatography with hydride generation atomic absorption spectrometry (HPLC/HG-AAS) via a flow-injection interface is presented. After arsenic species separation by anion-exchange displacement chromatography the compounds are on-line reduced to their corresponding hydrides and detected by atomic absorption spectrometry. Detection limits range from 1.1 (TMAO) to 2.6 microg/L (As(V)). The method has been applied to determine arsenic species in the urine of a volunteer before and after consumption of seafood as well as to analyse certified reference urine samples for their arsenic species content.

Rice blast fungus isolates were collected in Kyushu to investigate resistance to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D). In 2001, failure of control of rice blast was reported in the Saga prefecture, where MBI-Ds have been used since 1998. At that time, the distribution of resistant isolates was mainly limited to that area. However, in 2002, resistant isolates were detected in all prefectures of Kyushu. DNA fingerprinting analysis showed that the mutation causing resistance to MBI-Ds had arisen independently in each area. These data suggest that resistant isolates may occur in any area and become dominant under continuous selection pressure for MBI-Ds. Nevertheless, resistant strains can be controlled by reductase inhibitors of melanin biosynthesis (MBI-R) or commercial rice seed disinfectants.

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.

The task of inventing and developing active ingredients with useful biological activities requires a search for novel chemical substructures. This process may trigger the discovery of whole classes of chemicals of potential commercial interest. Similar biological effects can often be achieved by completely different compounds. However, compounds within a given structural family may exhibit quite different biological activities depending on their interactions with different intracellular proteins like enzymes or receptors. By varying the functional groups and structural elements of a lead compound, its interaction with the active site of the target protein, as well as its physicochemical, pharmacokinetic, and dynamic properties can be improved. In this context, the introduction of fluorine into active ingredients has become an important concept in the quest for a modern crop protection product with optimal efficacy, environmental safety, user friendliness, and economic viability. Fluorinated organic compounds represent an important and growing family of commercial agrochemicals. A number of recently developed agrochemical candidates represent novel classes of chemical compounds with new modes of action; several of these compounds contain new fluorinated substituents. However, the complex structure-activity relationships associated with biologically active molecules mean that the introduction of fluorine can lead to either an increase or a decrease in the efficacy of a compound depending on its changed mode of action, physicochemical properties, target interaction, or metabolic susceptibility and transformation. Therefore, it is still difficult to predict the sites in a molecule at which fluorine substitution will result in optimal desired effects.

The neonicotinoid insecticide imidacloprid is the most important insecticide in hop cultivation in Germany. A laboratory study was undertaken to investigate its systemic properties and translaminar bioavailability in hop leaves. Radiolabelled [methylene-14C]imidacloprid was applied either alone or in combination with different additives onto leaves of several hop varieties. Uptake and translocation were evaluated 1 and 7 days after foliar application under greenhouse conditions. The uptake of imidacloprid into hop leaves was most pronounced in the first 24 h after application and only negligible amounts were taken up after this period. Significant differences in the quantitative uptake occurred when imidacloprid was combined with additives, such as Amulsol, Genapol C-100, Hasten and LI 700. The uptake of imidacloprid applied without additives was less than 10% 7 days after application, whereas the combination with LI 700 provided 70-80% uptake. Genapol C-100 and Amulsol induced considerable phytotoxicity at the application site. Comparing hop varieties revealed differences up to twofold in foliar penetration of imidacloprid. The translaminar and acropetal bioavailability of imidacloprid foliarly applied to hop leaves was determined by a laboratory bioassay using the damson hop aphid, Phorodon humuli (Schrank). Significantly higher mortality was observed with laboratory formulations containing imidacloprid and the additive LI 700. In contrast to these results from systemic tests, contact mortality at the application site was constantly high over the testing period of 7 days, highlighting the importance of this mode of entry for aphid intoxication.

Fungi have an absolute requirement for K+, but K+ may be partially replaced by Na+. Na+ uptake in Ustilago maydis and Pichia sorbitophila was found to exhibit a fast rate, low Km, and apparent independence of the membrane potential. Searches of sequences with similarity to P-type ATPases in databases allowed us to identify three genes in these species, Umacu1, Umacu2, and PsACU1, that could encode P-type ATPases of a novel type. Deletion of the acu1 and acu2 genes proved that they encoded the transporters that mediated the high-affinity Na+ uptake of U. maydis. Heterologous expressions of the Umacu2 gene in K+ transport mutants of Saccharomyces cerevisiae and transport studies in the single and double Deltaacu1 and Deltaacu2 mutants of U. maydis revealed that the acu1 and acu2 genes encode transporters that mediated high-affinity K+ uptake in addition to Na+ uptake. Other fungi also have genes or pseudogenes whose translated sequences show high similarity to the ACU proteins of U. maydis and P. sorbitophila. In the phylogenetic tree of P-type ATPases all the identified ACU ATPases define a new cluster, which shows the lowest divergence with type IIC, animal Na+,K(+)-ATPases. The fungal high-affinity Na+ uptake mediated by ACU ATPases is functionally identical to the uptake that is mediated by some plant HKT transporters.

To investigate the role of stored and neosynthesized mRNAs in seed germination, we examined the effect of alpha-amanitin, a transcriptional inhibitor targeting RNA polymerase II, on the germination of nondormant Arabidopsis seeds. We used transparent testa mutants, of which seed coat is highly permeable, to better ascertain that the drug can reach the embryo during seed imbibition. Even with the most permeable mutant (tt2-1), germination (radicle protrusion) occurred in the absence of transcription, while subsequent seedling growth was blocked. In contrast, germination was abolished in the presence of the translational inhibitor cycloheximide. Taken together, the results highlight the role of stored proteins and mRNAs for germination in Arabidopsis and show that in this species the potential for germination is largely programmed during the seed maturation process. The alpha-amanitin-resistant germination exhibited characteristic features. First, this germination was strongly slowed down, indicating that de novo transcription normally allows the synthesis of factor(s) activating the germination rate. Second, the sensitivity of germination to gibberellic acid was reduced 15-fold, confirming the role of this phytohormone in germination. Third, de novo synthesis of enzymes involved in reserve mobilization and resumption of metabolic activity was repressed, thus accounting for the failure in seedling establishment. Fourth, germinating seeds can recapitulate at least part of the seed maturation program, being capable of using mRNAs stored during development. Thus, commitment to germination and plant growth requires transcription of genes allowing the imbibed seed to discriminate between mRNAs to be utilized in germination and those to be destroyed.

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.

Animal tetraspanins are membrane proteins controlling cell adhesion, morphology and motility. In fungi, the tetraspanin MgPls1 controls an appressorial function required for the penetration of Magnaporthe grisea into host plants. An orthologue of MgPLS1, BcPLS1, was identified in the necrotrophic fungal plant pathogen Botrytis cinerea. We constructed a Bcpls1::bar null mutant by targeted gene replacement. Bcpls1::bar is not pathogenic on intact plant tissues of bean, tomato or rose, but it infects wounded plant tissues. Both wild type and Bcpls1::bar differentiate appressoria on plant and artificial surfaces, a process involving an arrest of polarized growth, apex swelling and its cell wall reinforcement. Although wild-type appressoria allowed the penetration of the fungus into the host plant within 6-12 h, no successful penetration events were observed with Bcpls1::bar, suggesting that its appressoria are not functional. An eGFP transcriptional fusion showed that BcPLS1 was specifically expressed in conidia, germ tubes and appressoria during host penetration. Our results indicate that BcPLS1 is required for the penetration of B. cinerea into intact host plants. The defect in pathogenicity of Bcpls1::bar also demonstrates that functional B. cinerea appressoria are required for a successful penetration process. As Bcpls1::bar and Mgpls1 Delta::hph penetration defects are similar, fungal tetraspanins are likely to be required for an essential appressorial function widespread among fungi.

Potent new inhibitors of NADH:ubiquinone oxidoreductase (complex I) have been designed, with the help of molecular modelling, by hybridisation of known complex I inhibitors with inhibitors of cytochrome c oxidoreductase. The most interesting compound was the chromone 7 which was a selective inhibitor of complex I (IC(50) 15 nM) and showed acaricidal activity against spider mites.

Isoxaflutole, the active ingredient in BALANCE WDG and BALANCE PRO corn herbicides and a co-formulant with the herbicide flufenacet in the product EPIC, is readily degraded in soil and water to RPA 202248 alpha(-(cyclopropylcarbonyl)-2-(methyvlsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile). Because RPA 202248 is responsible at the molecular level for isoxaflutole's herbicidal activity it is important to understand the environmental behavior of the degradation product. Laboratory studies suggest that RPA 202248 is stable to hydrolysis and photolysis in aqueous systems and hence poses a possible environmental concern. As part of a program of work towards understanding the actual field situation, an outdoor microcosm study was carried out. Over the course of the 29-day study, residues remained predominantly in the aqueous phase. A slow but steady degradation of RPA 202248 was observed leading to the formation of RPA 203328 (2-methylsulfonyl-4-trifluoromethylbenzoic acid), which has no herbicidal activity. The half-life of RPA 202248 was calculated to be 103 days. These findings indicate that aqueous degradation should be considered as a potential route of dissipation when assessing the fate of RPA 202248 in large scale impounded water bodies, such as ponds, lakes, or reservoirs in the Mid-West Corn Belt.

Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay for phase-2 of the international validation exercise within the Organization for Economic Cooperation and Development (OECD). The chemicals studied included the antiandrogens finasteride (FIN) and procymidone (PRO) and the androgen agonist 17alpha-methyltestosterone (MT). Castration of sexually immature Sprague-Dawley rats was performed between post-natal days 42 and 46 whilst dosing of the chemical over 10 days was performed between post-natal days 53 and 67. Rats were co-treated with testosterone propionate (TP) for the antiandrogenic activity evaluation. The endpoints examined for evaluation of the androgenic/antiandrogenic activity were changes in sex accessory tissue (SAT) weights supplemented with measurement of testosterone and luteinizing hormone (LH) levels at sacrifice. Changes in liver, adrenal, kidney and body weights were also monitored for general toxicity assessment. Statistically significant changes in the SAT weights were detected with the three chemicals tested. Hence, the rat Hershberger assay as defined by the OECD was demonstrated sensitive enough for the detection of the endocrine disrupting activity of the three reference chemicals evaluated.

Sulfur occurs in two major amino-acids, cysteine (Cys) and methionine (Met), essential for the primary and secondary metabolism of the plant. Cys, as the first carbon/nitrogen-reduced sulfur product resulting from the sulfate assimilation pathway, serves as a sulfur donor for Met, glutathione, vitamins, co-factors, and sulfur compounds that play a major role in the growth and development of plant cells. This sulfur imprinting occurs in a myriad of fundamental processes, from photosynthesis to carbon and nitrogen metabolism. Cys and Met occur in proteins, with the former playing a wide range of functions in proteins catalysis. In addition, the sulfur atom in proteins forms part of a redox buffer, as for glutathione, through specific detoxification/protection mechanisms. In this review, a survey of sulfur assimilation from sulfate to Cys, Met and glutathione is presented with highlights on open questions on their respective biosynthetic pathways and regulations that derived from recent findings. These are addressed at the biochemical and molecular levels with respect to the fate of Cys and Met throughout the plant-cell metabolism.

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.

In this paper, the rheological properties, microscopic appearance and macroscopic sedimentation behaviour of 147- and 482-nm polystyrene latices in HEC solutions, bearing different adsorbed poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) copolymers are presented and compared with previous results with a 67-nm latex (Langmuir 13 (1997) 2922). The ratio of the steric layer thickness to the particle radius varies from 1:5 to 1:40 for the three latex sizes covering a range of particle softness. The 147-nm latex showed gas, liquid and solid phases, including three phase coexistence with increasing concentrations of HEC. The solid phase was a viscoelastic gel that sedimented slowly and showed initially slow, then faster sedimentation rates for HEC concentrations close to the fluid-gel phase boundary. These properties depended on the adsorbed PEO-PPO-PEO copolymers; the fluid-gel phase boundary moved to lower HEC concentrations with increasing PEO chain length. Oscillatory shear measurements were sensitive to the floc network structure and showed the transition from the fluid to gel phases. The values for the elastic modulus in the gel region were independent of the PEO chain length in the stabilizer implying the presence of similar floc structures with each of the different PEO-PPO-PEO copolymers. Stress relaxation measurements gave long relaxation times (> 10(3) s) and showed that the suspensions were viscoelastic fluids with high zero shear viscosities. Higher values were obtained with longer PEO chain lengths. Extrapolated yield stress values showed stronger flocculation with increasing PEO chain length in the adsorbed stabilising layer. There was good correlation between the extrapolated yield stress and the sedimentation velocity, indicating that the collapse of the floc network is governed by the strength of the inter-particle bonds (for the same floc structure) and also with the long-time behaviour from the stress relaxation measurements. With the 482-nm latex there was less effect of the adsorbed copolymer both for the rheology and sedimentation behaviour, with the particles behaving more like hard spheres. The transition between hard and soft sphere behaviour, interpreted here as the ratio of the steric layer thickness to the particle radius at which the adsorbed stabilising layer starts to have an effect on the rheology and phase behaviour is estimated to be approximately 1:20.

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is a serious pest in many cropping systems world-wide and occurs in different biotypes. The most widespread one is the B-type, whereas the Q-biotype is nowadays still mostly restricted to Southern Spain. Neonicotinoid cross-resistance is known at a high level in Q-types from Spain and individual samples collected in Italy and Germany. Now we detected for the first time high neonicotinoid cross-resistance in a B-type from Israel. Target site resistance to imidacloprid using [(3)H]imidacloprid in nicotinic acetylcholine receptor (nAChR) binding assays could not be detected in any of these highly resistant strains. The impact of metabolizing enzymes such as esterases, glutathione S-transferases, and cytochrome P450-dependent monooxygenases in neonicotinoid resistance was studied biochemically with artificial substrates. Monooxygenase activity was increased 2-3-fold in moderately resistant strains (RF approximately 30) and even 5-6-fold in highly resistant strains (RF approximately 1,000). Only monooxygenase activity correlated with imidacloprid, thiamethoxam and acetamiprid resistance and, therefore, monooxygenases seem to be the only enzyme system responsible for neonicotinoid resistance in B. tabaci Q- and B-types. The oxidative degradation of imidacloprid in resistant Q-type strains could be confirmed by metabolism studies of [(14)C]imidacloprid in vivo. Five-hydroxy-imidacloprid could be detected as the only main metabolite. The insecticidal activity and binding affinity to nAChR of this compound was 10 times lower than imidacloprid itself in B. tabaci., (Copyright 2003 Wiley-Liss, Inc.)

The COSMO-RS method, a combination of the quantum chemical dielectric continuum solvation model COSMO with a statistical thermodynamics treatment for more realistic solvation (RS) simulations, has been used for the direct prediction of pKa constants of a large variety of 64 organic and inorganic acids. A highly significant correlation of r(2) = 0.984 with a standard deviation of only 0.49 between the calculated values of the free energies of dissociation and the experimental pKa values was found, without any special adjustment of the method. Thus, we have a theoretical a priori prediction method for pKa, which has the regression constant and the slope as only adjusted parameters. Such a method can be of great value in many areas of physical chemistry, especially in pharmaceutical and agrochemical industry. To our surprise, the slope of pKa vs ΔGdiss is only 58% of the theoretically expected value of 1/RTln(10). A careful analysis with respect to different contributions as well as a comparison with the work of other authors excludes the possibility that the discrepancy is due to weaknesses of the calculation method. Hence, we must conclude that the experimental pKa scale depends differently on the free energy of dissociation than generally assumed.

A 28-day oral gavage toxicity study in the rat with 17alpha-methyltestosterone was conducted as part of the international validation exercise on the modified Enhanced OECD Test Guideline 407 (Organisation for Economic Co-operation and Development, Paris). Special emphasis was placed on the endocrine mediated effects exerted by 17alpha-methyltestosterone, a potent androgen agonist. The test compound was administered daily by oral gavage for at least 28 days to groups of 7-week-old-Wistar rats. Dose levels were 0, 10, 40 and 200 mg/kg body weight per day for males and 0, 10, 100 and 600 mg/kg body weight per day for females. In addition, and outside the remit of the enhanced protocol, testosterone levels in males, oestradiol levels in females and luteinizing hormone (LH) levels in both sexes were measured, to provide a broader profile on the hormonally mediated effects of 17alpha-methyltestosterone. Furthermore, stage-specific quantification of Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL)-labeled germ cells (apoptotic germ cells) in the seminiferous tubules was also performed, in an effort to demonstrate the precise stages in the spermatogenic cycle 17alpha-methyltestosterone exerts its effect. In this study, the most critical additional parameters contained in the Enhanced OECD Test Guideline 407 for the detection of endocrine disruption were considered to be the histopathological assessment and organ weight data of endocrine-related tissues. Beyond the scope of this validation exercise, an increase in apoptosis in specific germ cell types was detected using the TUNEL assay in male rats treated at 200 and 40 mg/kg.

The (S,S,S,R,S,R)-configurated cyclohexadepsipeptides (CHDPs) represent novel enniatin derivatives with strong in vivo activity against the parasitic nematode Haemonchus contortus Rudolphi in sheep. 2D NMR spectroscopic analysis revealed for the major conformation the asymmetric conformer, containing a cis-amide bond between C(alpha) protons of neighbouring 2-hydroxy-(S)-carboxylic acid and N-methyl-(S)-amino acid. The absolute configuration of the novel CHDPs was determined by X-ray crystallography. A correlation between the major conformer and its anthelmintic activity was found. Here, we report on a simple total synthetic pathway for this particular type of CHDPs.

The damson hop aphid, Phorodon humuli (Schrank) (Hemiptera: Aphididae), is one of the most important sucking pests of many hop-growing areas world-wide. In this study we determined the efficacy of several insecticides against strains collected throughout the year 2001. All strains were collected in different hop gardens in the Hallertau (Bavaria), Germany, the largest hop-growing area of the world. First of all we established a leaf dip bioassay, carried out using six-well tissue culture plates and appropriate for monitoring susceptibility against imidacloprid, oxydemeton-methyl, cyfluthrin, amitraz, pymetrozine and pirimicarb. Four of these compounds, imidacloprid, cyfluthrin, pymetrozine and amitraz, are currently registered for the control of sucking pests in German hop gardens and are useful against P. humuli. The leaf-dip bioassay system turned out to be very reliable and robust. Ten P. humuli strains were collected in May 2001 and maintained in the laboratory to assess their resistance to the different insecticides in comparison with two laboratory reference strains (H2 and H5). Using diagnostic concentrations, resistance monitoring for imidacloprid and cyfluthrin was investigated during July and August 2001 on 53 populations from 30 sites around the Hallertau, an area of ca 2500 km2. Resistance to diagnostic concentrations (LC95 for reference strains) of imidacloprid, amitraz and pymetrozine was not detected in any strain received in 2001, but late-season (August) populations seemed to respond more heterogeneously than those collected mid-season (July). Overall composite mean mortalities to diagnostic concentrations of imidacloprid (13 mg litre(-1)) in collections from May, July and August were 95 (+/-2.5), 98 (+/-2.3) and 87 (+/-5.9)%, respectively. Moderate resistance to pyrethroids was observed in all strains collected in May and August using a diagnostic concentrations of 10 mg litre(-1) cyfluthrin (LC95 of the susceptible reference strain H5). Slight to moderate resistance to diagnostic concentrations of oxydemeton-methyl and pirimicarb was observed in some, but not all, strains collected early season. The results are discussed in terms of the implemention of hop aphid resistance management strategies in German hop-cultivation areas.

The main objective of this 28-day oral gavage toxicity study in the rat was to investigate which of the current and/or additional parameters of the OECD Test Guideline 407 would reliably and sensitively detect the endocrine-mediated effects of the nonsteroidal antiestrogen tamoxifen. In addition, as this study was performed using two subgroups of five animals of each sex run concurrently, it enabled an assessment of the intralaboratory reproducibility while also assessing the potential value of using ten animals of each sex per group instead of using the standard five animals of each sex per group stipulated by the current guideline. Tamoxifen was administered daily by gavage to groups of 7-week-old Wistar rats for at least 28 days at dose levels of 5, 30, or 200 microg/kg body weight. Additional parameters specified in the enhanced OECD Test Guideline 407 were spermatozoa enumeration and morphology of the cauda epididymis, hormonal analysis of the thyroid-stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4) levels, monitoring of the estrous cycle during week 4 of treatment to ensure females were in diestrus on the day of terminal sacrifice, organ weight of ovary, uterus, thyroid gland, prostate gland (ventral and dorsolateral parts), seminal vesicles with coagulation glands and pituitary gland, and microscopic investigation of the pituitary gland, vagina, mammary gland, seminal vesicles with coagulating glands, epididymis, and prostate gland (ventral and dorsolateral parts). Overall, 200 microg/kg per day was considered to be the Maximum Tolerated Dose (MTD) in both sexes, resulting in a marked reduction of body weight gain, together with slight effects on clinical signs, hematology, plasma chemistry, and microscopic changes in some endocrine tissues. Five micrograms per kilogram per day represented the No Observed Adverse Effect Level (NOAEL) in males and the No Observed Effect Level (NOEL) in females. At the intermediate dose level (30 microg/kg per day), the current OECD Test Guideline 407 was appropriate to detect the specific endocrine-related changes induced by tamoxifen in females, based on the histopathology findings observed in the ovary and the uterus. The additional parameters which were found to be changed in females (thyroid hormone levels, ovary and uterus weights, and histopathology of vagina) provided supplementary information further confirming tamoxifen-mediated endocrine effects. In males, when data from the current Test Guideline 407 were considered at the intermediate dose level, specific endocrine effects were only indicated on the basis of the histopathology findings observed in the prostate gland. The additional parameters examined which were found to be changed (prostate gland and seminal vesicle weights, and histopathology of seminal vesicle and mammary gland) were necessary to confirm the specific tamoxifen-mediated endocrine effects. Hence, amongst the additional parameters contained in the enhanced OECD Test Guideline 407, organ weights and histopathological examination of endocrine-related organs were the most helpful in confirming the detection of tamoxifen-mediated endocrine effects. The reproducibility evaluation showed that a group size of five animals of each sex consistently allowed the detection of endocrine effects with the current Test Guideline in both sexes at the high dose level and in females at the intermediate dose level. Doubling the animal number from five to ten of each sex per dose level did not notably increase the sensitivity of detection of endocrine-mediated effects.

In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.

The derivatization reaction of the mycotoxin moniliformin with 1,2-diamino-4,5-dichlorobenzene was previously introduced to improve distinctly the sensitivity of an assay applying high-performance liquid chromatography prior to fluorescence detection. In the course of the implementation of this derivatization approach into a liquid chromatographic/mass spectrometric method, an unexpected derivatization product has now been discovered by mass spectrometry. In order to elucidate its structure, detailed investigations with liquid chromatography/tandem mass spectrometry and liquid chromatography coupled on-line with NMR spectroscopy were performed. These studies give evidence for a heterocyclic structure that has been formed by the loss of one water and one carbon monoxide molecule. A reasonable mechanism for this derivatization reaction is proposed., (Copyright 2003 John Wiley & Sons, Ltd.)

We investigated whether the allocation of rodenticide baiting points to specific structural elements would result in complete rat eradication on livestock farms, as opposed to assigning the baiting points only to places where there were obvious signs of rat activity. The goal was to establish an effective rodent-control program that is easy for untrained persons to conduct.Rat-control strategies were examined on 25 farms in Velen (Muensterland), Germany, where an average of 20% of trapped rats were resistant for bromadiolone according to a blood-clotting response (BCR) test. All farms were investigated for signs of rat activity prior to and after the control measure. Differences in the percentage level of farmer compliance in setting up the baiting points as prescribed were analysed for each type of baiting point and in total, and were compared between the group of farms which achieved complete rat eradication and those which did not. Farms achieving complete eradication had an average of 81% compliance with prescribed control plans, whereas a significantly lower compliance level of only 51% was recorded on farms that did not achieve eradication. A >/=75% level of implementation of the control plan always resulted in complete control success. The new method of bait-point allocation was incorporated into a self-explanatory computer program, which was verified to be effective during a rat-control campaign in the restricted area after an outbreak of classical swine fever near Soltau in northern Germany, in July 2001. This program, which is available on the Internet, enables the creation of individualised rat-control plans, including complete documentation of the control measure.

Field exposure of small mammals to fungicide-treated wheat seed was investigated over three weeks following drilling on fields near York, United Kingdom. Seed consumption by small mammals trapped on and immediately adjacent to the drilled fields was quantified by measuring the amount of seed in the stomach. In addition, exposure to one seed-treatment, fluquinconazole, was quantified by measuring residues of the fungicide in the stomach, liver, and intestine. The wood mouse, Apodemus sylvaticus, was the dominant species caught on the fields and the only species found to have consumed measurable quantities of seed. Voles, Microtus agrestis and Clethrionomys glareolus, were caught in small numbers, almost exclusively in the field hedge, and showed no evidence of having consumed seed. Stomach-contents analysis revealed that more than 80% of animals trapped in the hedge adjacent to the field had consumed no wheat seed, whereas 98% had consumed less than 10% (by stomach volume). Ninety percent of animals trapped on the field had consumed seed, although 90% of these animals had less than 20% seed in the stomach. Residues of the fungicide in the stomach, intestine, and liver were lower than would be expected for the amount of seed consumed, possibly because of dehusking of the seeds by mice. The relevance of these findings when assessing exposure (and risk) posed by seed treatments to wild mammals is discussed.

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays. This system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo.

Different insect neuropeptides (helicokinins, tachykinin-related and allatoregulating peptides) were investigated with regard to their myostimulatory effects using whole-gut preparations isolated from fifth instar Heliothis virescens larvae. The experiments demonstrated that representatives of all three peptide families are able to induce and amplify gut contractions in this species in a dose-dependent manner. Structure-activity studies (alanine scan, D-amino acid scan and truncated analogues) with the helicokinin Hez-K1 supported the finding, that the core sequence for biological activity of kinins is the amidated C-terminal pentapeptide (FSPWG-amide). Similar investigations with insect tachykinin isolated from Leucophaea madera (Lem-TRP1) revealed that the minimum sequence evoking a physiological gut response in H. virescens is the amidated hexapeptide (GFLGVR-amide), which represents the conserved amino acid sequence for Leucophaea TRPs in general. The peptide concentration causing a half-maximal gut contraction (EC(50)) for Lem-TRP1 was about 26 nM. Although the potency of Lem-TRP1 was 9-fold lower compared with Hez-KI (EC(50): 3 nM), the maximal tension of the gut obtained with Lem-TRP1 was 1.7-fold higher compared with Hez-KI. The EC(50) of Manduca sexta allatotropin (Mas-AT; 79 nM) was of lowest potency among all three peptides tested. In a pharmacological study, co-incubation experiments with Lem-TRP1, Hez-KI or Mas-AT and compounds interfering with signal transduction pathways were employed to investigate the mode of action of the myotropic effects of these peptides. Cadmium and the protein kinase C (PKC) inhibitor tamoxifen attenuated the contractile effects of all three peptides tested. The data suggest that in the gut muscle of H. virescens the myotropic peptides bind to G-protein-coupled receptors that cause contraction by promoting the entry of extracellular calcium mediated by a PKC involved pathway.

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.

The susceptibility to several insecticides of 16 and 8 strains of Myzus persicae Sulzer and Aphis gossypii Glover, respectively, received from different European countries in 2001 was investigated. Most of the strains were derived from places known for their aphid resistance problems to conventional insecticides before imidacloprid was introduced. In many regions and agronomic cropping systems imidacloprid has been an essential part of aphid control strategies for a decade, and therefore the susceptibility of aphid populations to imidacloprid using FAO-dip tests and diagnostic concentrations in a leaf-dip bioassay was checked. Additional insecticides tested were cyfluthrin (chemical class: pyrethroid), pirimicarb (carbamate), methamidophos and oxydemeton-methyl (organophosphates). Diagnostic concentrations (LC99-values of reference strains) for each insecticide were established by dose response analysis using a new leaf-disc dip bioassay format in 6-well tissue culture plates. Virtually no resistance to imidacloprid in any of the field-derived populations of M. persicae and A. gossypii was detected. In contrast, strong resistance was found to pirimicarb and oxydemeton-methyl, and to a lesser extent also to cyfluthrin. Two strains of A. gossypii exhibited reduced susceptibility to imidacloprid when tested directly after collection. However, after maintaining them for six weeks in the laboratory, the aphids were as susceptible as the reference strain. The diagnostic concentration of methamidophos did not reveal any resistance in M. persicae, but did so in four strains of A. gossypii.

The heterozygous p53 knockout mouse model was used to assess whether vascular tumors noted in a 2-year carcinogenicity study in CD-1 mice with carbaryl were induced through a genotoxic mechanism. This knockout mouse model was selected for carbaryl because of the high sensitivity of this model to genotoxic events and its low spontaneous incidence of tumors until 9-12 months of age. Carbaryl was administered continuously via the diet to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000 and 4000 ppm for 180 days. Histopathological examinations revealed no evidence of carbaryl-induced neoplasms of any type. In particular, no neoplastic or preneoplastic changes were noted in the vascular tissue of any of the organs examined. Only neoplasms, recognized as those that occur spontaneously in untreated mice of this strain, were sporadically observed in a few animals from the intermediate dose groups with no evidence of a dose- or treatment-related effect. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) of carbaryl for neoplastic changes in male mice was 4000 ppm (around 716 mg/kg body weight/day). We conclude: (1) carbaryl does not appear to be a genotoxic carcinogen at least in male mice; (2) if the vascular tumors observed in the CD-1 mice are treatment-related, they could have been induced by a non-genotoxic mechanism; (3) the response in transgenic animals may provide useful complementary results to better assess carbaryl's potential genotoxic hazard to humans.

The N-methylated amidoxime analogues of the cyclic octadepsipeptide PF1022A represent novel derivatives with activity against Trichinella spiralis Owen and Nippostrongylus brasiliensis Lane in vitro and against parasitic nematodes in mice and sheep. Some of them show better activity against Hymenolepis nana Siebold, Heterakis spumosa Schneider and Heligmosomoides polygyrus Dujardin in mice than the natural product PF1022A. In particular an improved efficacy against Haemonchus contortus Rudolphi and Trichostrongylus colubriformis Giles in sheep compared to the potent cyclic octadepsipeptide PF1022A and its mono-thionated derivative has been observed. Here we report on a specific modification at the N-methyl amide linkage by using the mono-thionated PF1022A, resulting in novel anthelmintically active backbone analogues of PF 1022A.

Knowledge on plant genomes has progressed during the past few years. Two plant genomes, those of Arabidopsis thaliana and rice, have been sequenced. Our present knowledge of synteny also indicates that, despite plasticity contributing to the diversity of the plant genomes, the organization of genes is conserved within large sections of chromosomes. In parallel, novel plant transformation systems have been proposed, notably with regard to plastid transformation and the removal of selectable marker genes in transgenic plants. Furthermore, a number of recent works considerably widen the potential of plant biotechnology.

Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.