Juvenile hormone (JH) esterase activity but not JH epoxide hydrolase activity is downregulated in larval Adoxophyes honmai following nucleopolyhedroviruses infection

Juvenile hormones (JHs) and ecdysteroids are critical insect developmental hormones. JH esterase (JHE) and JH epoxide hydrolase (JHEH) are JH-selective enzymes that metabolize JH and thus regulate the titer of JH. Baculoviruses are known to alter host endocrine regulation. The nucleopolyhedroviruses, AdhoNPV and AdorNPV, are known to have slow and fast killing activity against Adoxophyes honmai (Lepidoptera: Tortricidae), respectively. Here we found that when penultimate (4th) instar A. honmai are inoculated with AdhoNPV or AdorNPV, the mean survival time is 9.7 and 8.2 days, respectively. The larvae molted once but did not pupate. The AdhoNPV- or AdorNPV-infected larvae did not show a dramatic increase in JHE activity as was found in mock-infected larvae, instead they showed a marked decrease in JHE activity. In contrast, both viral infections had no effect on JHEH activity. In order to further characterize the JHE activity, the JHE-coding sequence of A. honmai (ahjhe) was cloned and confirmed to encode a biologically active JHE. Quantitative real-time PCR analysis of ahjhe expression in 4th and 5th instar A. honmai revealed that AdhoNPV and AdorNPV are able to reduce ahjhe expression levels.     

Variabilité génétique entre les populatiosn de Speonomus zophosinus

Genetic variability was examined in six troglobitic populations of Speonomus zophosinus which were limited to a very small geographical area. Results indicate a high genetic variation and a high degree of heterozygosity (H 0.43). The genetic variability observed between the populations reveals the existence of two genetically heterogeneous groups (one upstream, one downstream. There are significant differences in the frequency of the six alleles of the locus Est 3 found in these two groups.     

A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth,Plutella xylostella

Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI = 5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.     

Phylogeny,classification and metagenomic bioprospecting of microbial acetyl xylan esterases

Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products.Although one-third of all characterized CEs within CAZy families 1⿿7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks.The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.     

Allelic isozyme variants in the mexican axolotl (Ambystoma mexicanum) as potential markers for developmental experiments

Four isozyme systems were surveyed in our laboratory-bred colony of axolotls (Ambystoma mexicanum) to determine whether there were elecrophoretic variants that could be used as markers in developmental experiments. For malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and phosphoglucomutase (PGM), the best separations were obtained by isoelectric focussing on polyacrylamide slab gels, whereas for soluble esterases (Est), conventional polyacrylamide gel electrophoresis was used. The patterns for both MDH and LDH were consistent with two-locus models, but no variation was obtained. The results for PGM support a single-locus model with two alleles that are expressed codominantly in heterozygotes. There is also evidence for a third, null allele. The pgm gene maps approximately 24 map units from its centromere. The majority of the animals tested produced four esterase bands. We propose that each is controlled by a separate locus. One of the bands, Est-3, is absent in some animals. The results of various crosses support the proposition that these animals are homozygous for a null allele. The est-3 gene is distant from its centromere.     

Polymorphism of transferrin and esterase in Alaskan wolves: evidence of close molecular homology with the dog

By the use of isoelectric focusing in polyacrylamide gels serum samples from 146 Alaskan wolves were studied with regard to transferrin (Tf) and esterase (ArE) polymorphism, comparing the phenotypic band patterns with those of selected Norwegian dogs. The study revealed Tf and ArE polymorphisms in the wolf with phenotypic band patterns being indistinguishable from the corresponding ones in dogs. This suggests the occurrence of the same two common Tf alleles in the wolf as in the dog. In the ArE system the results are consistent with the occurrence of three alleles which also occur in dogs whereas a fourth allele, so far not seen in dogs, is seen in Alaskan wolves.     

5-(4-Acetoxy-1-butinyl)-2,2′-bithiophene:acetate esterase from Tagetes patula

From the aerial parts of Tagetes patula, an enzyme with high substrate specificity, namely 5-(4-acetoxy-1-butinyl)-2,2′-bithiophene:acetate esterase, was partly purified. The enzyme has a MW of 67000 (±5000), pH optimum of 7.5 and its activity is affected considerably in the presence of bovine serum albumin (BSA). BSA at a concentration of 5 mg/ml in the reaction mixture prevents substrate polymerization.     

Improved insecticidal efficacy of a recombinant baculovirus expressing mutated JH esterase from Manduca sexta

Juvenile hormone esterase (JHE), a member of the carboxylesterase family (EC 3.1.1.1), metabolizes JH that is found in juvenile insects. A highly conserved amphipathic alpha helix is found on the surface of known JHEs. This helix is implicated in receptor-mediated binding and endocytosis of JHE by the pericardial cells resulting in the clearance of JHE activity from the hemolymph. In this study, Lys-204 and Arg-208 of the amphipathic alpha helix of the JHE of Manduca sexta (MsJHE) were mutated to histidine residues generating MsJHE-HH. Pharmacokinetic studies following the injection of MsJHE-HH into the hemocoel of larval M. sexta, Heliothis virescens, and Agrotis ipsilon indicated that MsJHE-HH and wild type MsJHE are cleared at similar rates. The infectivity (lethal concentration and lethal time) of a recombinant baculovirus, AcMsJHE-HH, expressing MsJHE-HH was not significantly different than that of a recombinant baculovirus, AcMsJHE, expressing MsJHE in first instars of H. virescens and A. ipsilon. However, the mass of AcMsJHE-HH-infected larvae was 40–50% lower than that of larvae infected with AcMsJHE, and 70–90% lower than that of wild type AcMNPV-infected larvae.     

Characterization of three-dimensional rat central nervous system culture maturation,with applications to monitor cholinergic integrity

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H₂O₂, glutamate, or amyloid β(1–42); each resulted in diminished AChE activity. H₂O₂ exposure resulted in nuclear fragmentation. Glutamate and amyloid β(1–42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.     

Genetics of esterases in Drosophila. VII. Genetic control of the activity level of juvenile hormone (JH)-esterase and heat resistance in D. virilis at high temperatures

The heat-resistant subline 147S was obtained in Drosophila virilis by selecting for viability individuals of heat-sensitive stock 147. It was shown that in the heat-treated 147S pupae the activity of juvenile hormone (JH)-esterase is decreased and, consequently, the titer of juvenile hormone is increased compared with those in the control pupae. These changes are consistent with those observed earlier for resistant stock 101. Heat-resistant stocks 101 and 147S were crossed with heat-sensitive stock 147, whose heat-treated larvae show earlier activation and higher activity of JH-esterase than control larvae. The viability and electrophoretic esterase patterns were analyzed in the F₁ and F₂ hybrids at different temperatures. It was found that the F₁ hybrid is resistant to the effect of high temperature and its activity level of JH-esterase is lower compared with controls. In the F₂ hybrid, there was a 3:1 segregation of viability and a 1:2:1 segregation of the activity level of JH-esterase at high temperatures. It is concluded that the activity level of JH-esterase and heat resistance in D. virilis are monogenically controlled at high temperatures.