Isolation of juvenile hormone esterase and its partial cDNA clone from the beetle, Tenebrio molitor.

Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme has been targeted for development of biologically-based insecticides. JHE was partially purified from the beetle, Tenebrio molitor, using a transition state analog as the affinity ligand. Two forms of JHE were characterized by activity analysis, isoelectric focusing, two-dimensional SDS-PAGE and N-terminal sequence analysis. The esterase is associated with two proteins of sizes 71 and 150 kDa, both of which are active on JH III. A partial cDNA clone for the enzyme was isolated based on the sequence of N-terminal and internal peptides. Its sequence indicates that JHE from T. molitor and Heliothis virescens may have a common origin.

Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta.

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activity). As expected, Ms-JHEH was localized in the microsomal fraction with a molecular mass of approximately 50 kDa. Ms-JHEH showed a substrate and inhibitor spectrum similar to the wild type JHEH isolated from eggs of M. sexta. Its enzymatic activity was the highest for Juvenile Hormone III. Ms-JHEH hydrolyzed several trans-epoxides faster than cis-epoxides. A putative hydroxyl-acyl enzyme intermediate was isolated suggesting a catalytic mechanism of Ms-JHEH similar to the mammalian EHs.

Recombinant baculoviruses for insect control.

Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

Synthesis of new carboxylesterase inhibitors and evaluation of potency and water solubility.

Carboxylesterases are essential enzymes in the hydrolysis and detoxification of numerous pharmaceuticals and pesticides. They are vital in mediating organophosphate toxicity and in activating many prodrugs such as the chemotherapeutic agent CPT-11. It is therefore important to study the catalytic mechanism responsible for carboxylesterase-induced hydrolysis, which can be accomplished through the use of potent and selective inhibitors. Trifluoromethyl ketone (TFK)-containing compounds are the most potent esterase inhibitors described to date. The inclusion of a thioether moiety beta to the carbonyl further increased TFK inhibitor potency. In this study, we have synthesized the sulfone analogues of a series of aliphatic and aromatic substituted thioether TFKs to evaluate their potency and solubility properties. This structural change shifted the keto/hydrate equilibrium from <9% hydrate to >95% hydrate, forming almost exclusively the gem-diol. These new compounds were evaluated for their inhibition of carboxylesterase activity in three different systems, rat liver microsomes, commercial porcine esterase, and juvenile hormone esterase in cabbage looper (Trichoplusia ni) hemolymph. The most potent inhibitor of rat liver carboxylesterase activity was 1,1,1-trifluoro-3-(decane-1-sulfonyl)-propan-2,2-diol, which inhibited 50% of the enzyme activity (IC(50)) at 6.3 +/- 1.3 nM and was 18-fold more potent than its thioether analogue. However, the sulfone derivatives were consistently poorer inhibitors of porcine carboxylesterase activity and juvenile hormone esterase activity, with IC(50) values ranging from low micromolar to millimolar. The compound 1,1,1-trifluoro-3-(octane-1-sulfonyl)-propan-2,2-diol was shown to have a 10-fold greater water solubility than its thioether analogue, 1,1,1-trifluoro-3-octylsulfanyl-propan-2-one (OTFP). These novel compounds provide further evidence of the differences between esterase orthologs, suggesting that additional development of esterase inhibitors may ultimately provide a battery of ortholog and/or isoform selective inhibitors analogous to those available for other complex enzyme families with overlapping substrate specificity.

Visualization of individual reovirus particles by low-temperature, high-resolution scanning electron microscopy.

We used low-temperature, high-resolution scanning electron microscopy (cryo-HRSEM) to visualize surface structures on individual reovirus particles. Both intact virions and two forms of subvirion particles--infectious subvirion particles and cores--were examined, and despite some distortion of particles during specimen preparation and viewing in the microscope, the images obtained by cryo-HRSEM exhibited a level of interpretable detail not routinely achieved by other methods without image averaging. Cryo-HRSEM images of discrete reovirus particles were used to characterize and confirm features of the outer protein capsid of this virus by comparison with image reconstructions previously derived from cryotransmission electron microscopy. Distinct surface features attributable to each of the four outer-capsid proteins were identified. In addition, cryo-HRSEM images confirmed that significant changes occur on the surfaces of individual reovirus particles during disassembly and entry of cells and that the reovirus outer capsid is organized as a left-handed T = 13 icosahedron. Several unique capabilities and potential uses suggest that cryo-HRSEM has a place alongside other, more established methods for molecular characterizations of virus particles.

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.