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ABSTRACT: Trehalase is involved in the control of trehalose concentration, the main blood sugar in insects. Here, we describe the molecular cloning of the cDNA encoding for the soluble form of the trehalase from the midge larvae of Chironomus riparius, a well-known bioindicator of the quality of freshwater environments. Molecular cloning was achieved through multiple alignment of Diptera trehalase sequences, allowing the synthesis of internal homology-based primers; the complete open reading frame(ORF) was subsequently obtained through RACE-PCR(where RACE is rapid amplification of cDNA ends). The cDNA contained the 5' untranslated region (UTR), the 3' UTR including a poly(A) tail and the ORF of 1,725 bp consisting of 574 amino acid residues with a predicted molecular mass of 65,778 Da. Recombinant trehalase was successfully expressed in Escherichia coli as a His-tagged protein and purified on Ni-NTA affinity chromatography. Primary structure analysis showed a series of characteristic features shared by all insect trehalases, while three-dimensional structure prediction yielded the typical glucosidase fold, the two key residues involved in the catalytic mechanism being conserved. Production of recombinant insect trehalases opens the way to structural characterizations of the catalytic site, which might represent, among others, an element for reconsidering the enzyme as a target in pest insects' control.
Archives of Insect Biochemistry and Physiology 07/2012; 81(2):77-89. · 1.36 Impact Factor
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ABSTRACT: A small set of nojirimycin- and pyrrolidine-based iminosugar derivatives has been synthesized and evaluated as potential inhibitors of porcine and insect trehalases. Compounds 12, 13 and 20 proved to be active against both insect and porcine trehalases with selectivity towards the insect glycosidase, while compounds 10, 14 and 16 behaved as inhibitors only of insect trehalase. Despite the fact that the activity was found in the micromolar range, these findings may help in elucidating the structural features of this class of enzymes of different origin, which are still scarcely characterised.
Beilstein Journal of Organic Chemistry 01/2012; 8:514-21. · 2.52 Impact Factor
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ABSTRACT: A preparation of a membrane-bound trehalase from the larvae of the midge Chironomus riparius (Diptera: Chironomidae) was obtained by detergent solubilization, ion-exchange chromatography and concanavalin A affinity chromatography. Trehalase was purified 1080-fold to a specific activity of 75 U mg(-)(1). The initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a K(m) of 0.48 +/- 0.04 mM. Catalytic efficiency was maximal at pH 6.5. The activity was highly inhibited by mono- and bicyclic iminosugar alkaloids such as (in order of potency) casuarine (IC(50) = 0.25 +/- 0.03 microM), deoxynojirimycin (IC(50) = 2.83 +/- 0.34 microM) and castanospermine (IC(50) = 12.7 +/- 1.4 microM). Increasing substrate concentration reduced the inhibition. However, in the presence of deoxynojirimycin, Lineweaver-Burk plots were curvilinear upward. Linear plots were obtained with porcine trehalase. Here, we propose that deoxynojirimycin inhibits the activity of trehalase from C. riparius according to a ligand exclusion model. Inhibition was further characterized by measuring enzyme activity in the presence of a series of casuarine and deoxynojirimycin derivatives. For comparison, inhibition studies were also performed with porcine trehalase. Results indicate substantial differences between midge trehalase and mammalian trehalase suggesting that, in principle, inhibitors against insect pests having trehalase as biochemical targets can be developed.
Glycobiology 09/2010; 20(9):1186-95. · 3.58 Impact Factor
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ABSTRACT: Two novel casuarine-6-alpha-D-glucoside analogues, as well as the parent compound, were synthesized and tested as inhibitors towards Chironomus riparius, mammalian pig kidney and Escherichia coli trehalases. Their potent and selective activity is promising for the development of new insecticides.
Chemical Communications 04/2010; 46(15):2629-31. · 6.17 Impact Factor
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ABSTRACT: The impact of pesticides on macrobenthos communities was studied on two aquatic environments in Northern Italy; River Meolo, a site exposed to agricultural pollution, and River Upper Livenza a low pollution reference site. Colonisation of artificial substrates was compared throughout the productive season. The relevance of multiple environmental stressors other than pesticides (e.g. oxygen depletion) was also assessed. Biochemical indicators (enzyme and metabolite biomarkers) were measured on selected organisms. Biomarker results (acetylcholinesterase, glutathione S-transferase, and napthylacetate esterase), as well as community structure patterns, revealed a significant difference between the two rivers. Cause-effect relationships between results and multiple stress factors were discussed.
Aquatic Toxicology 09/2008; 89(1):1-10. · 3.76 Impact Factor
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ABSTRACT: Changes in enzyme activities, metabolite concentrations, and membrane transport activity underlying the Chironomus riparius larvae adaptive response to anoxia were investigated. Trehalose, malate, and aspartate degradation and alanine accumulation were recorded. During anoxia exposure, there was a boost of antioxidant defenses as shown by an increase of the specific activity of the enzymes catalase, glutathione-S-transferase, glutathione peroxidase, glutathione-synthase, malic enzyme, and NADP-dependent isocitrate dehydrogenase. The ratio, glutathione reduced over glutathione oxidized, decreased. Except for alanine and catalase, the parameters return to their basal value when larvae are transferred to normoxic conditions. To test whether antioxidant defenses had protective effects on membrane functionality, L-leucine uptake into brush border membrane vesicles and membrane lipid peroxidation was measured. No difference between membranes prepared from larvae exposed to anoxia and control larvae was found. The amino acid alanine, when present inside the vesicles, trans-stimulated leucine uptake. This effect could represent a mechanism to stimulate amino acid uptake and catabolism in vivo when free alanine concentration increases during hypoxic periods.
Archives of Insect Biochemistry and Physiology 09/2007; 65(4):181-94. · 1.36 Impact Factor
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ABSTRACT: Chironomus riparius Meigen were exposed to three different insecticides, the organophosphorous fenitrothion and the carbamates carbaryl and carbofuran (0, 1, 10, and 100 microg/L) for 24h as fourth-instar larvae. Acetylcholinesterase (AChE), naphtylacetate esterase (NAE), p-nitrophenylacetate esterase (PNPAE), glutathione-S-transferase (GST), and a number of metabolites (alanine, pyruvate, lactate, trehalose, aspartate, oxalacetate) were measured to determine which was the most valuable biochemical biomarker of exposure. AChE activity was significantly reduced by all three insecticides, PNPAE by fenitrothion, carbofuran and carbaryl, whereas NAE activity was stimulated by carbaryl and unaffected by fenitrothion and carbofuran. Metabolites analysis revealed a strong accumulation of alanine in larvae exposed to fenitrothion, but not in larvae exposed to carbamates. This accumulation was accompanied by a significant increase of lactate and a significant decrease of pyruvate and trehalose. No variations were observed with carbofuran and carbaryl. No change of aspartate concentration was detected. We conclude that the association of alanine accumulation with a significant inhibition of AChE activity can be used as a valuable biochemical biomarker of exposure.
Ecotoxicology and Environmental Safety 04/2007; 66(3):326-34. · 2.29 Impact Factor
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ABSTRACT: Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects.
Archives of Insect Biochemistry and Physiology 12/2006; 63(3):110-22. · 1.36 Impact Factor
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ABSTRACT: The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.
Archives of Insect Biochemistry and Physiology 03/2004; 55(2):90-101. · 1.36 Impact Factor
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ABSTRACT: A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.
Journal of insect physiology 06/2002; 48(5):585-592. · 2.24 Impact Factor
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ABSTRACT: Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12–18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch. Insect Biochem. Physiol. 48:190–198, 2001. © 2001 Wiley-Liss, Inc.
Archives of Insect Biochemistry and Physiology 11/2001; 48(4):190 - 198. · 1.36 Impact Factor
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ABSTRACT: A net absorption of sodium ions and ofl-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroachBlabera gigantea. Both sodium and amino acid net fluxes were abolished by the haemolymphatic addition of the Na–K ATPase inhibitor ouabain, at a concentration 1 mM. A purified fraction of brush border membranes, prepared from the midgut tissue by Ca-precipitation, was used to investigate the occurrence of a cotransport system in the luminal membrane of the cockroach enterocyte. An inwardly directed Na gradient (100 mM outside the vesicles, 0 mM inside) drives the uphill movement of phenylalanine into the vesicular space, whilst other monovalent cations fail to induce the concentrative uptake of the amino acid. Moreover, the amino acid uptake seems to be dependent on the transmembrane potential, since inwardly directed gradients of different sodium salts determine a decreasing rate of phenylalanine uptake in agreement with the presumptive permeabilities of Na counterions. These data suggest the presence of a Na-phenylalanine cotransport system located on the brush border membrane ofB. gigantea midgut.
Journal of Comparative Physiology B 06/1986; 156(4):549-556. · 1.97 Impact Factor
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ABSTRACT: Experiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K+-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K+-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatK
m
for histidine and lysine increased 10-fold and three-fold, respectively, whereasV
max was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.
Journal of Membrane Biology 01/1985; 88(1):45-53. · 1.81 Impact Factor
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ABSTRACT: In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when Kout>Kin and Kout=Kin.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.
Journal of Membrane Biology 09/1984; 81(3):233-240. · 1.81 Impact Factor
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ABSTRACT: The luminal membrane of larval midgut cells is the site of action of insecticidal delta-endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+-amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ permeability of brush border membrane vesicles prepared from mammalian small intestine,
FEBS Letters.
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ABSTRACT: L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, α-, β-, γ-, δ-, ε-amino acids, primary amines, α-amino alcohols, monocarboxylic organic acids and α-ketoacids. Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of δ- and ε-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed.
Insect Biochemistry and Molecular Biology.
