Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).
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1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase. (+info)
Altered properties of neuronal sodium channels associated with genetic resistance to pyrethroids.
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Genetic resistance to pyrethroid insecticides involves nervous system insensitivity linked to regulatory and structural genes of voltage-sensitive sodium channels. We examined the properties and relative density of sodium channels in central neurons of susceptible and pyrethroid-resistant (Pyr-R) insects that were homozygous for the amino acid substitution V421M in the I-S6 transmembrane segment. Pyr-R sodium channels show approximately 21-fold lower sensitivity to the synthetic pyrethroid permethrin and a approximately 2-fold increased sensitivity to the alpha-scorpion toxin LqhalphaIT. Pyr-R channels also exhibit altered gating properties, including a approximately 13 mV positive shift in voltage-dependent activation and approximately 7 mV positive shift in steady-state inactivation. Consistent with these changes in gating behavior, Pyr-R central neurons are less excitable, as evidenced by an approximately 11 mV elevation of action potential threshold. No differences in sodium channel density are evident. The altered properties of Pyr-R sodium channels provide a plausible molecular basis for nervous system insensitivity associated with pyrethroid resistance. (+info)
Ion binding and permeation through the lepidopteran amino acid transporter KAAT1 expressed in Xenopus oocytes.
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1. The transient and steady-state currents induced by voltage jumps in Xenopus oocytes expressing the lepidopteran amino acid co-transporter KAAT1 have been investigated by two-electrode voltage clamp. 2. KAAT1-expressing oocytes exhibited membrane currents larger than controls even in the absence of amino acid substrate (uncoupled current). The selectivity order of this uncoupled current was Li+ > Na+ approximately Rb+ approximately K+ > Cs+; in contrast, the permeability order in non-injected oocytes was Rb+ > K+ > Cs+ > Na+ > Li+. 3. KAAT1-expressing oocytes gave rise to 'pre-steady-state currents' in the absence of amino acid. The characteristics of the charge movement differed according to the bathing ion: the curves in K+ were strongly shifted (> 100 mV) towards more negative potentials compared with those in Na+, while in tetramethylammonium (TMA+) no charge movement was detected. 4. The charge-voltage (Q-V) relationship in Na+ could be fitted by a Boltzmann equation having V of -69 +/- 1 mV and slope factor of 26 +/- 1 mV; lowering the Na+ concentrations shifted the Q-V relationship to more negative potentials; the curves could be described by a generalized Hill equation with a coefficient of 1.6, suggesting two binding sites. The maximal movable charge (Qmax) in Na+, 3 days after injection, was in the range 2.5-10 nC. 5. Addition of the transported substrate leucine increased the steady-state carrier current, the increase being larger in high K+ compared with high Na+ solution; in these conditions the charge movement disappeared. 6. Applying Eyring rate theory, the energy profile of the transporter in the absence of organic substrate included a very high external energy barrier (25.8 RT units) followed by a rather deep well (1.8 RT units). (+info)
O-glycosylation potential of lepidopteran insect cell lines.
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The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line. (+info)
Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity.
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Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements. (+info)
Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.
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DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi. (+info)
Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.
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In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context. (+info)
Global protein synthesis shutdown in Autographa californica nucleopolyhedrovirus-infected Ld652Y cells is rescued by tRNA from uninfected cells.
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Global protein synthesis arrest occurs in Autographa californica nucleopolyhedrovirus (AcNPV)-infected Ld652Y cells at late times postinfection (p.i.). A Lymantria dispar nucleopolyhedrovirus gene, hrf-1, precludes this protein synthesis arrest. We used in vitro translation assays to characterize the translation defect. Cell-free lysates prepared from uninfected Ld652Y cells, AcNPV-infected cells harvested at early times p.i., and cells infected with vAchrf-1, a recombinant AcNPV bearing hrf-1, all supported translation. Lysates prepared from AcNPV-infected Ld652Y cells at late times p.i. did not support translation, but activity was restored by adding small RNA species from mock-, vAchrf-1- (24 or 48 h p.i.), and AcNPV- (6 h p.i. ) infected cells. Small RNA species (24 and 48 h p.i.) from AcNPV-infected cells did not rescue translation. Assays of RNA species further fractionated by ion exchange chromatography demonstrated that tRNA rescued translation. Although specific defective tRNA species were not revealed by comparative two-dimensional gel analysis, analysis of (32)P-labeled tRNAs showed a reduction in de novo synthesis of small RNA isolated from AcNPV-infected cells compared with mock- and vAchrf-1-infected cells. This study suggests a mechanism of translation arrest involving defective or depleted tRNA species in AcNPV-infected Ld652Y cells. (+info)