Leafrollers in apple IPM under regimes based on Bacillus thuringiensis, on diflubenzuron, or on epofenonane

Three selective insecticides for leafroller control were evaluated as part of research efforts for refinement of Integrated Pest Management (IPM) in apple orchards in the Netherlands. Population levels, injury to fruits and degree of parasitism of leafrollers were studied in 0.5–1 ha plots of an experimental orchard. Plots were sprayed during 5 consecutive years with either Dimilin^® (diflubenzuron, a chitine synthesis inhibitor), a strain of Bacillus thuringiensis or epofenonane, a juvenile hormone analogue. Dipel and Dimilin were not sufficiently effective in reducing population levels and degree of fruit injury. Hard to control pests in Dipel-treated plots were especially Pandemis heparana, Spilonota ocellana and Laspeyresia pomonella and in Dimilin treated plots P. heparana and Adoxophyes orana.Epofenonane could not be evaluated properly because of interference by immigration of moths from nearby untreated plots.

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Résumé L'étude a porté sur la protection, par 3 insecticides sélectifs, de vergers attaqués par des tordeuses. Ce travail constitue une part des travaux menés aux Pays-bas pour améliorer la lutte intégrée dans les vergers.Nous avons étudié les niveaux des populations de tordeuses, leurs dégâts sur fruits, ainsi que les taux de parasitisme, dans un verger expérimental divisé en parcelles de 0,5 à I ha.Pendant 5 années consécutives, les différentes parcelles ont subi des pulvérisations de Dimiline (diflubenzuron), inhibiteur de la synthèse de chitine, de Dipel (B. thuringiensis), et enfin d'épophénonane, un analogue de l'hormone juvénile.Le Dipel et la Dimiline ont présenté peu d'efficacité dans la réduction des populations et du niveau des dégâts. Les insectes particulièrement difficiles à contrôler sont, respectivement, dans les vergers traités par le Dipel: Pandemis heparana (Denn. et Schiff.), Spilonota ocellana (F.) et Laspeyresia pomonella (L.), et dans ceux traites par la Dimiline: P. heparana et Adoxophyes orana (F.v.R). Nous n'avons pas pu évaluer l'effet de l'épophénonane d'une fâçon satisfaisante, en raison des réinfestations par des papillons venant de terrains voisins hébergeant des populations élevées de tordeuses.

     

Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.     

Synthesis of gamma-monodentate and beta, gamma-bidentate CrITP. Isolation and characterization of the two chelation isomers and the individual diastereomers of beta, gamma-bidentate CrITP

The two chelation isomers of CrITP, gamma-monodentate and beta, gamma-bidentate CrITP, as well as the diastereomers of beta, gamma-bidentate CrITP were synthesized, isolated, and characterized. Synthesis of these complexes was done using pH titration methods similar to that described by Cleland [W.W. Cleland, Methods Enzymol. 87, 159 (1982)], and separation of the two chelation isomers was accomplished with DEAE-sephadex A-25 using 0-0.3 N linear HCl gradient. Diastereomer separation (analytical and preparative scales) of beta, gamma-bidentate CrITP using reverse-phase high-performance liquid chromatography, and then analysis of the diastereomers with circular dichroism spectroscopy, shows four diastereomers that exist as two pairs of mirror-image isomers, similar to the four diastereomers of beta, gamma-bidentate CrATP as presented by Dunaway-Mariano and Cleland [D. Dunaway-Mariano and W.W. Cleland, Biochemistry 19, 1496 (1980)]. Reverse-phase high-performance liquid chromatography analysis of gamma-monodentate CrITP shows the presence of two major peaks, both of which convert to beta, gamma-bidentate CrITP upon incubation at pH 6.0 for 1 hr.     

2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions

Rate constants for the hydrolysis of acetyl-TPP were measured between pH values of 2.5 and 7.5 and plotted as log kobs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649]. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pKa of 4.73 within acetyl-TPP that is remote from the acetyl group, the amino-pyrimidine ring, which is protonated below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP [Gruys, K.J., Halkides, C.J., & Frey, P.A. (1987) Biochemistry 26, 7575-7585] have facilitated the isolation and identification of [1-14C]acetyl-TPP from acid-quenched enzymatic reaction mixtures at steady states. [1-14C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E1). The pH-rate profile for hydrolysis of [1-14C]-acetyl-TPP isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the 14C-labeled enzymic species.     

Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism’s intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-β-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form β-ketovaleryl-CoA. This activity has generally been attributed to the β-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a β-ketothiolase from R. eutropha that is capable of forming β-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of β-hydroxyvalerate derived from propionyl-CoA. We also report an additional β-ketothiolase, designated BktC, that probably serves as a secondary route toward β-hydroxyvalerate production.Polyhydroxyalkanoates (PHAs) are a class of naturally occurring polymers which serve as a carbon and energy reserve in numerous bacterial species. Ralstonia eutropha (formerly designated Alcaligenes eutrophus [41]) produces the homopolymer poly(β-hydroxybutyrate) (PHB) and, when provided with propionate in the feedstock, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). R. eutropha is used commercially to produce PHBV, which is a biodegradable thermoplastic.The PHB biosynthetic pathway requires three enzymatic activities: a β-ketothiolase (PhbA), an NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase (PhbB) and a PHB synthase (PhbC). The first step in production of the homopolymer PHB is catalyzed by β-ketothiolase which condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Formation of the copolymer PHBV requires the additional condensation of acetyl-CoA with propionyl-CoA to form β-ketovaleryl-CoA (Fig. ​(Fig.1).1). Subsequently, the acetoacetyl-CoA and β-ketovaleryl-CoA are converted into a polymer by the activities of the reductase and synthase. The genes encoding these proteins in R. eutropha reside in an operon which has been well characterized (10, 21, 22, 31, 37). Open in a separate windowFIG. 1Pathway for production of PHBV from acetyl-CoA and propionyl-CoA. β-Ketothiolase performs the condensation reactions to generate either acetoacetyl-CoA or β-ketovaleryl-CoA. These are reduced by acetoacetyl-CoA reductase (PhbB) and polymerized by PHB synthase (PhbC).The substrate specificities of these three enzymes are reportedly adequate for production of PHBV copolymer (7–9), but propionate-fed Escherichia coli harboring the R. eutropha phb operon produces essentially PHB homopolymer (35). Moreover, PHBV copolymer can be produced in E. coli after induction of the fatty acid β-oxidation complex, which contains a β-ketothiolase with broad substrate specificity (26, 27, 35). These data suggest that the R. eutropha PHB pathway is capable of producing copolymer, but only in the context of a second β-ketothiolase with broad substrate specificity.R. eutropha is known to produce at least two β-ketothiolases (7), and at least two distinct plasmid clones which express β-ketothiolase have been isolated from R. eutropha (37). In this work, we analyzed the substrate specificity of the PhbA β-ketothiolase and demonstrated that this enzyme catalyzes thiolysis of β-ketovaleryl-CoA very poorly. We determined that R. eutropha expresses at least two β-ketothiolases in addition to PhbA and that these additional enzymes, which we designate BktB and BktC, efficiently utilize β-ketovaleryl-CoA. We also report the isolation and characterization of bktB (β-ketothiolase B), which encodes the BktB β-ketothiolase required for efficient production of PHBV in R. eutropha.     

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.     

Expression of fungal diacylglycerol acyltransferase2 genes to increase kernel oil in maize

Maize (Zea mays) oil has high value but is only about 4% of the grain by weight. To increase kernel oil content, fungal diacylglycerol acyltransferase2 (DGAT2) genes from Umbelopsis (formerly Mortierella) ramanniana and Neurospora crassa were introduced into maize using an embryo-enhanced promoter. The protein encoded by the N. crassa gene was longer than that of U. ramanniana. It included 353 amino acids that aligned to the U. ramanniana DGAT2A protein and a 243-amino acid sequence at the amino terminus that was unique to the N. crassa DGAT2 protein. Two forms of N. crassa DGAT2 were tested: the predicted full-length protein (L-NcDGAT2) and a shorter form (S-NcDGAT2) that encoded just the sequences that share homology with the U. ramanniana protein. Expression of all three transgenes in maize resulted in small but statistically significant increases in kernel oil. S-NcDGAT2 had the biggest impact on kernel oil, with a 26% (relative) increase in oil in kernels of the best events (inbred). Increases in kernel oil were also obtained in both conventional and high-oil hybrids, and grain yield was not affected by expression of these fungal DGAT2 transgenes.     

Parasites of fishes in the recently inundated ephemeral Lake Liambezi,Namibia

Lake Liambezi forms the periodic connection between the upper Zambezi, Kwando and Okavango rivers. A full parasitological assessment was conducted on 86 fish, representing 14 species in six families sampled in August 2011. Parasite diversity was low and dominated by species with complex life cycles involving intermediate hosts. Most prevalent were larval nematodes (Contracaecum sp.) infecting 12 and Trypanasoma sp. infecting nine of the 14 host species. The intra-erythrocytic parasite Babesiosoma mariae was found in the blood of Coptodon rendalli and Oreochromis andersonii with prevalence of 50% and 60%, respectively. The host-specific monogenean Annulotrema hepseti was recorded only from H. cuvieri with a prevalence of 100%. Notable absences were the copepod and branchiuran parasites that have direct lifecycles and usually occur in high prevalence and abundance in the region. Because parasites with direct life cycles can only be transported into the lake on the host fish, their absence suggests limited immigration of infected fishes into the lake. This suggests that internal recruitment dominates over immigration in the fish population dynamics in Lake Liambezi.     

Augmentation of natural cell activity in tumor-bearing and normal mice by MVE-2

Summary Mice bearing advanced Lewis lung carcinoma were found to have significantly decreased natural killer (NK) cell activity in spleen and blood. The same pattern of lowered spontaneous NK cell activity was observed in nude mice with advanced human colon carcinoma LS 174 and in C3H mammary tumor virus-positive mice that spontaneously developed mammary adenocarcinomas. Maleic anhydride divinyl ether (MVE-2) usually augments NK cell activity in normal mice. We found that the lower level of spontaneous NK cell activity in tumor-bearing mice could be boosted by a single injection of MVE-2; however, this response was much weaker than that observed in age-matched normal mice. Multiple treatments with MVE-2 which are known to induce hyporesponsiveness to further augmentation of NK cell activity in spleen and blood of normal mice, also produced NK cell hyporesponsiveness in the spleen, bone marrow, and blood of tumor-bearing mice.Wladyslaw Budzynski is a guest researcher in the Preclinical Screening Laboratory from the Institute of Immunology and Experimental Therapy, Czerska Street 12, Wroclaw, Poland     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.