Functional analysis of novel sulfotransferases in the silkworm Bombyx mori

Sulfoconjugation plays a vital role in the detoxification of xenobiotics and in the metabolism of endogenous compounds. In this study, we aimed to identify new members of the sulfotransferase (SULT) superfamily in the silkworm Bombyx mori. Based on amino acid sequence and phylogenetic analyses, two new enzymes, swSULT ST1 and swSULT ST2, were identified that appear to belong to a distinct group of SULTs including several other insect SULTs. We expressed, purified, and characterized recombinant SULTs. While swSULT ST1 sulfated xanthurenic acid and pentachlorophenol, swSULT ST2 exclusively utilized xanthurenic acid as a substrate. Based on these results, and those concerning the tissue distribution and substrate specificity toward pentachlorophenol analyses, we hypothesize that swSULT ST1 plays a role in the detoxification of xenobiotics, including insecticides, in the silkworm midgut and in the induction of gametogenesis in silkworm ovary and testis. Collectively, the data obtained herein contribute to a better understanding of SULT enzymatic functions in insects.     

Mixed function oxidases and herbivore polyphagy: the devil's advocate position

Abstract. 1. Evidence used to support the theory that mixed function oxidases (MFOs) are important in the survival of polyphagous herbivores is briefly reviewed. This evidence includes data on patterns of variation in MFO activity among species and among developmental stages within species. It also includes data on induction of MFO activity by plant compounds and metabolism of plant cornpounds by MFOs.
2. It is argued that the evidence presently available does not offer strong support of the theory because key pieces of information are lacking. Evidence which tends to refute the theory is reviewed and discussed.
3. Experiments are proposed which could more rigorously test the theory.     

Phosphate compounds as iron chelators in animal cell cultures

Summary We have studied the capacity of a number of phosphate compounds to act in the double role as a phosphate source and a detoxifier of ferric chloride hydroxo compounds, i.e. as Fe(III) chelators. The tested compounds were: orthophosphate, trimetaphosphate, α-glycerophosphate, β-glycerophosphate, phytic acid, and phosphorylcholine; the test organism the ciliate protozoonTetrahymena thermophila, an animal cell; and the nutrient medium was synthetic, consisting solely of low-molecular-weight compounds. We assessed growth rates of cells in two experimental series. First, phosphate-starved cells were exposed to the tested phosphate compound as the only phosphate source and the ferric chloride concentrations were varied stepwise from 0 to 1000μM. Second, we offered the cells orthophosphate as a phosphate source and selected phosphate compounds as chelators. The cell growth results allow the following conclusions: orthophosphate, trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent phosphate sources; trimetaphosphate, α-glycerophosphate, β-glycerophosphate, and phytic acid are excellent Fe(III) chelators; of the tested compounds trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent in the double role as a phosphate source and a ferric chloride hydroxo detoxifier, i.e. as a Fe(III) chelator.     

Expression of biotransformation genes in woodrat (Neotoma) herbivores on novel and ancestral diets: identification of candidate genes responsible for dietary shifts

The ability of herbivores to switch diets is thought to be governed by biotransformation enzymes. To identify potential biotransformation enzymes, we conducted a large-scale study on the expression of biotransformation enzymes in herbivorous woodrats ( Neotoma lepida ). We compared gene expression in a woodrat population from the Great Basin that feeds on the ancestral diet of juniper to one from the Mojave Desert that putatively switched from feeding on juniper to feeding on creosote. Juniper and creosote have notable differences in secondary chemistry, and thus, should require different biotransformation enzymes for detoxification. Individuals from each population were fed juniper and creosote diets separately. After the feeding trials, hepatic mRNA was extracted and hybridized to laboratory rat microarrays. Hybridization of woodrat samples to biotransformation probes on the array was 87%, resulting in a total of 224 biotransformation genes that met quality control standards. Overall, we found large differences in expression of biotransformation genes when woodrats were fed juniper vs. creosote. Mojave woodrats had greater expression of 10× as many biotransformation genes as did Great Basin woodrats on a creosote diet. We identified 24 candidate genes that may be critical in the biotransformation of creosote toxins. Superoxide dismutase, a free radical scavenger, was also expressed to a greater extent by the Mojave woodrats and may be important in controlling oxidative damage during biotransformation. The results are consistent with the hypothesis that biotransformation enzymes limit diet switching and that woodrats in the Mojave have evolved a unique strategy for the biotransformation of creosote toxins.     

Modes of action of fraxinellone against the tobacco budworm, Heliothis virescens

Fraxinellone significantly reduced the relative growth rate, food consumption rate as well as the efficiency of conversion of ingested food into biomass of Heliothis virescens when incorporated into artificial diets at concentrations of 4.31 × 10⁻⁵ mol/L and above. After being fed with fraxinellone-treated diets for 24 h, the larval midguts of H. virescens possess significantly lower activities of α-amylase and non-specific proteases and higher activities of cytochrome P450s. In vitro , the compound did not inhibit the activities of α-amylase and non-specific proteases extracted from the larval midguts. Clear evidence of post-ingestive toxicity of fraxinellone to midgut cells was observed under an electron microscope. The modes of action of the compound against insects were discussed.     

Biological degradation of explosives and chemical agents

Hazardous energetic organo-nitro compounds are found as contaminants in many environments. A series of nitro aromatics, nitrate esters and nitro amines, all characteristic of this class, has been studied for their susceptibility to biological transformation. Biotransformation pathways for each of these compounds have been identified and are summarized. Implications for these findings in light of current contamination issues is discussed. The detoxification of organophosphate chemical agents focuses on the investigation of organophosphate degrading enzymes from bacteria. Certain of these enzymes, active both in solution and when immobilized onto a solid surface, are very successful in hydrolyzing and detoxifying various organophosphate chemical agents. The relationship of this research to the critical concerns of the agricultural industry regarding detoxification of organophosphorous pesticides is discussed.     

Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilised on a nylon membrane chemically grafted with glycidyl methacrylate (GMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GLU) were used as spacer and bifunctional coupling agent, respectively. Quinol was used as substrate.

To know how the immobilisation procedures affected the enzyme reaction rate the catalytic behaviour of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged from the experimental data: (i) the narrower pH-activity profile of the insoluble enzyme in comparison to that of the soluble counterpart; (ii) the increase of the affinity of the immobilised enzyme for its substrate.

The behaviour of the catalytic membrane was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increases with the increase of the temperature difference. These results have been discussed in the frame of reference of the process of thermodialysis driving thermodiffusive transmembrane substrate fluxes, which add to the diffusive ones.

The advantages of the catalytic process carried out under non-isothermal conditions have been thrown in relief through the evaluation of the reduction of the production times and of the percentage increases of the enzyme activity.     

NADP+-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification

An NADP⁺-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (K_M 33 µM, k_cat 405 min⁻¹), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic–δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.