Evaluation of insecticidal and genotoxic effects of imidacloprid and acetochlor in Drosophila melanogaster

Two pesticides, the insecticide imidacloprid and the herbicide acetochlor, were evaluated for their insecticidal and genotoxic effects in Drosophila melanogaster . Their insecticidal effects were assessed by calculating LC₅₀ values after acute or chronic exposure of larvae and adults to different concentrations of the test compounds. After acute exposure, the LC₅₀ of imidacloprid was 7.59 × 10⁻⁵ M for larvae and 1.43 × 10⁻⁴ M for adults, and after chronic exposure, it was 2.67 × 10⁻⁵ M and 6.09 × 10⁻⁵ M for larvae and adults, respectively. On the contrary, the herbicide acetochlor showed no acute or chronic insecticidal effect against either larvae or adults, even at very high concentrations (8 × 10⁻³ M). For the evaluation of genotoxic properties of the two pesticides, the Somatic Mutation and Recombination Test in D. melanogaster was used. Our results suggest that neither imidacloprid nor acetochlor exhibits mutagenic or recombinogenic activity at applicable concentrations.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Cytokinin-binding proteins from tobacco callus share homology with osmotin-like protein and an endochitinase

To study the signal transduction of cytokinins, we characterized cytokinin-binding proteins (CBPs) isolated from tobacco callus Nicotiana tabacum. Two high-affinity CBPs, CBP1 and CBP2, were isolated from the soluble fraction of tobacco callus BY-2 cells by anion exchange chromatography on a DEAE-cellulose column and affinity chromatography on a benzyladenine (BA)-linked Sepharose 4B column. Cytokinin-binding activity was determined by the equilibrium dialysis method. The degree of purification of CBP1 and CBP2 was 270 and 600-fold, respectively. These proteins had molecular masses of 34 kDa and 26 kDa, and to bind benzyladenine (BA) with dissociation constants (Kd) of 8.9 x 10(-6) M and 1.1 x 10(-6) M, respectively. Binding of BA to CBP2 was inhibited by zeatin and kinetin but not by adenine, adenosine, ATP or IAA. The optimum pH for binding of BA to CBP1 and CBP2 was approximately pH 6.5 and 7.5, respectively. CBP1 showed significant homology (90%) with endochitinase and CBP2 with osmotin-like protein (OLP). These findings and the results of immunoblotting analysis and cytokinin-binding assay of recombinant OLP indicated that CBP2 is OLP, a stress protein.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Baseline toxicity of emamectin and spinosad to Helicoverpa armigera (Lepidoptera: Noctuidae) for resistance monitoring

Acute toxicity studies of emamectin and spinosad against Helicoverpa armigera revealed that the pest is highly susceptible to both the insecticides. The median lethal dose (LD₅₀) of emamectin is 3.86 × 10⁻³ µg per larva. The median lethal concentrations (LC₅₀) of emamectin and spinosad were found to be 0.09 and 2.94 ppm, respectively. The discriminating doses were fixed based on the LC₉₅ of the susceptible population of H. armigera as 0.80 ppm for emamectin and 10 ppm for spinosad. Resistance was not observed when the discriminating doses of emamectin and spinosad were applied on field-collected populations of H. armigera from two intensive cotton growing areas, Coimbatore and Madurai, India.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

VIP down-regulates the inflammatory potential and promotes survival of dying (neural crest-derived) corneal endothelial cells ex vivo: necrosis to apoptosis switch and up-regulation of Bcl-2 and N-cadherin

The neuropeptide vasoactive intestinal peptide (VIP) is anti-inflammatory and protective in the immune and nervous systems, respectively. This study demonstrated in corneal endothelial (CE) cells injured by severe oxidative stress (1.4 mM H₂O₂) in bovine corneal organ cultures that VIP pre-treatment (0, 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M; 15 min), in a VIP concentration-dependent manner, switched the inflammation-causing necrosis to inflammation-neutral apoptosis (showing annexin V-binding, chromatin condensation, and DNA fragmentation) and upheld ATP levels in a VIP antagonist (SN)VIPhyb-sensitive manner, while up-regulated mRNA levels of the anti-apoptotic Bcl-2 and the differentiation marker N-cadherin in a kinase A inhibitor-sensitive manner. As a result, VIP, in a concentration-dependent and VIP antagonist-sensitive manners, promoted long-term CE cell survival. ATP levels, a determining factor in the choice of apoptosis versus necrosis, measured after VIP pre-treatment and 0.5 min post-H₂O₂ were 39.6 ± 3.3, 50.8 ± 6.2, 60.1 ± 4.8, and 53.6 ± 5.3 pmoles/μg protein (mean ± SEM), respectively ( p  < 0.05, anova ). VIP treatment alone concentration-dependently increased levels of N-cadherin ( Koh et al. 2008 ), the phosphorylated cAMP-responsive-element binding protein and Bcl-2, while10⁻⁸ M VIP, in a VIP antagonist (SN)VIPhyb-sensitive manner, increased ATP level by 38% ( p  < 0.02) and decreased glycogen level by 32% ( p  < 0.02). VPAC1 (not VPAC2) receptor was expressed in CE cells. Thus, CE cell VIP/VPAC1 signaling is both anti-inflammatory and protective in the corneal endothelium.     

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

Volatile organic compound (VOC) emissions from tobacco ( Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O₃) were investigated using proton-transfer-reaction mass-spectrometry (PTR-MS) and gas chromatography mass-spectrometry (GC-MS) to find a quantitative reference for plants' responses to O₃ stress. O₃ exposures to illuminated plants induced post-exposure VOC emission bursts. The lag time for the onset of volatile C₆ emissions produced within the octadecanoid pathway was found to be inversely proportional to O₃ uptake, or more precisely, to the O₃ flux density into the plants. In cases of short O₃ pulses of identical duration the total amount of these emitted C₆ VOC was related to the O₃ flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C₆ product was emitted per five O₃ molecules taken up by the plant. A threshold flux density of O₃ inducing emissions of C₆ products was found to be (1.6 ± 0.7) × 10⁻⁸ mol m⁻² s⁻¹.     

Purification and characterization of phosphoglucomutase from peas

Phosphoglucomutase (EC 2.7.5.1) was isolated from pea seeds ( Pisum sativum L. cv. Grenadier) and purified to homogeneity as determined by sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The enzyme was purified by utilizing 25% polyethylene glycol 4000 precipitation, followed by Fractogel-diethyla-minoethyl (DEAE) 650. Fractogel-TSK HW-55(s). and high pressure liquid chroma-tography (HPLC)-(PEI) column chrornatography. The resulting enzyme had a specific activity of 157 units (mg protein)-1. a 152-fold increase over that of the crude plant extract. The molecular weight of the enzyme was 128 to 136 kDa. as determined by native-PAGE and column chromatography, and when it was subjected to SDS-PAGE analysis, it was found to be composed of two subunits having molecular weights ranging from 59 to 64 kDa. Upon SDS-PAGE analysis of a sample purified through HPLC-PEI chromatography. two bands of protein were found: one having a molecular weight of 64 kDa and the other 68 kDa. A pH optimum of 8.6 was found for the enzyme while it was also found that cysleine. Mg²⁺ and glucose 1.6-bisphosphate were necessary for optimal activity Histidine and imidazole only partially fulfilled the cysteine requirement. A 20-min preincubation period in the absence of glucose 1-phosphate was necessary for optimal activity of the enzyme. Without a preincubation period, there was a pronounced lag preceding the linear portion of the reaction as well as a reduction in the V_max. An analysis of the kinetics of the reaction showed K_m values ot 3.6 × 10⁻⁵ and 1.45 × 10⁻⁵ M for glucose 1-phosphate and glucose 1.6-bisphosphate. respectively. A K., of 7.3 × 10⁻⁵ M was obtained for MgCl₂.     

Dormancy breaking and frost resistance induction in apple embryos as related to changes in reserve and polar lipids

Detailed dose-response curves were obtained for cytokinin-induced retention of chlorophyll in detached leaves of barley, Hordeum vulgare L. cv. Møyar, over a wide range, 10⁻¹⁰– 2.5 × 10⁻⁴ M , of external concentrations of benzyladeninc or kinetin. The over-all shape of the curves was log-linear and the response was subsensitive. Benzyladenine was somewhat more effective than kinetin. In the curves there were pronounced local minima and maxima both at low, intermediate (10⁻⁷– 10⁻⁵ M ) and high concentrations. At low and high concentrations these deviations from the best smooth curves were reproducible, statistically significant and occurred at the same concentrations for benzyladenine and kinetin. In the intermediate range the deviations from the log-linear line were marked and statistically highly significant in some but not all experiments. The cause of the complexities in the dose response curve is not known, but multiphasic uptake of the cytokinins may be involved.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

Age and growth of Anguilla anguilla in the Camargue lagoons

Age and total length ( L _T) data from a 11 year monitoring of the Anguilla anguilla eel population of the Camargue lagoons (Rhône delta, southern France) were collected for glass, yellow and silver eels. Three distinct models were calibrated to describe the growth process of undifferentiated eels, females and males, respectively. Uncertainty of parameter estimates was evaluated by bootstrapping. Females were characterized by larger asymptotic body size ( L _T) than males (580 ± 50 v . 388 ± 13 mm) and faster growth, whilst the Brody growth coefficient was larger for males than for females (means ±  s . d . 3·00 10⁻³ ± 1·68 10⁻³ v . 1·73 10⁻³ ± 0·50 10⁻³). Sexual differentiation was estimated to begin at 204 ± 38 mm mean ±  s . d ., i.e . at the end of the second year in the lagoons, well before the L _T at which macroscopic differentiation became possible ( c . 300 mm). Males probably leave the lagoon or die (due to either natural or fishing mortality) within the first 3 years, whilst females can remain up to 5 years. Sexual differentiation and maturation have a major role in shaping the L _T structure of the population. The L _T and mass ( M ) data were fitted by allometric curves     . The calibration of distinct curves for data from different years indicated that the allometric coefficient a was subject to wider interannual fluctuations than the allometric exponent b . A negative correlation linked the average L _T and the allometric exponent ( r  = −0·58, P  < 0·01).     

Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leaves

A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent K _m and V _max for the peptide were 1.18 m m and 2.27 mmol pNA mg⁻¹ h⁻¹, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65–75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process.     

Meeting US biofuel goals with less land: the potential of Miscanthus

Biofuels from crops are emerging as a Jekyll & Hyde – promoted by some as a means to offset fossil fuel emissions, denigrated by others as lacking sustainability and taking land from food crops. It is frequently asserted that plants convert only 0.1% of solar energy into biomass, therefore requiring unacceptable amounts of land for production of fuel feedstocks. The C₄ perennial grass Miscanthus × giganteus has proved a promising biomass crop in Europe, while switchgrass ( Panicum virgatum ) has been tested at several locations in N. America. Here, replicated side-by-side trials of these two crops were established for the first time along a latitudinal gradient in Illinois. Over 3 years of trials, Miscanthus × giganteus achieved average annual conversion efficiencies into harvestable biomass of 1.0% (30 t ha⁻¹) and a maximum of 2.0% (61 t ha⁻¹), with minimal agricultural inputs. The regionally adapted switchgrass variety Cave-in-Rock achieved somewhat lower yields, averaging 10 t ha⁻¹. Given that there has been little attempt to improve the agronomy and genetics of these grasses compared with the major grain crops, these efficiencies are the minimum of what may be achieved. At this 1.0% efficiency, 12 million hectares, or 9.3% of current US cropland, would be sufficient to provide 133 × 10⁹ L of ethanol, enough to offset one-fifth of the current US gasoline use. In contrast, maize grain from the same area of land would only provide 49 × 10⁹ L, while requiring much higher nitrogen and fossil energy inputs in its cultivation.