Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of covalently bound myo-inositol in bovine erythrocyte acetylcholinesterase and porcine kidney alkaline phosphatase

Bovine erythrocyte acetylcholinesterase and porcine kidney alkaline phosphatase were purified to a homogeneous state. By using gas chromatography-mass spectrometry, we demonstrated the presence of covalently bound myo-inositol in these purified enzymes. The quantitative data suggest that one molecule of myo-inositol is bound to each subunit of these enzyme proteins. The covalently bound inositol was removed from these enzyme molecules by deamination with nitrous acid, suggesting the possibility that myo-inositol is directly bound to amino sugar.     

Linkage analysis of an acetylcholinesterase gene in the house fly Musca domestica (Diptera: Muscidae)

Linkage of an acetylcholinesterase (AChE) gene was detected in the house fly, Musca domestica L., by using the backcross method between a strain, aabys, that had a morphological multichromosomal marker on each of the five autosomes and a wild strain, LPR. Both strains were homozygous in this gene, and we used eight single nucleotide polymorphisms (SNPs) between them to distinguish the parental sequences in the backcrossed progeny, two of which resulted in the amino acid substitiutions common to the Drosophila and Aedes AChEs insensitive to organophosphates and carbamates. F, appeared to be a wild phenotype, and the AChE gene was heterozyous of aabys and LPR. In the backcross progeny, 32 (2(5)) phenotypes appeared, and 10 phenotypes with one wild or morphological marker were picked up for genotyping by the SNPs of AChE gene. A combination of the morphological markers and the SNPs revealed that the AChE structural gene is linked to autosome 2 in the house fly.     

Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells

Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala?→?Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 μg/mL) for 12 h, RBLBP of 5 μg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 μg/mL of mutant LBP and the BMECs only treated with 10 μg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 μg/mL of wild LBP and 10 μg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands.     

Interaction of membrane-bound and solubilized erythrocyte acetylcholinesterase with carbamates

Physostigmine and neostigmine methylsulphate are shown to be the most strong inhibitors of acetylcholine esterase of human erythrocytes. The action of baigon is less pronounced and pyrimor is characterized as the weakest inhibitor. No differences are found between the membrane-bound and solubilized acetylcholine esterase relative to their ability to be inhibited by these carbamates. The preliminary treatment of acetylcholine esterase with carbamates protects the enzyme from the subsequent inhibition by the organophosphoric inhibitor. A higher concentration (1.6-2.1 times) of physostigmine and pyrimor and lower (1.7-1.9 times) one of baigon and neostigmine methylsulphate are needed for protection of the soluble enzyme than of the membrane-bound enzyme.     

Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme

Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.     

The major histocompatibility complex and mating preferences in wild house mice (mus domesticus)

This study examined the relationship between the major histocompatibilitycomplex (MHC) genes and mate choice by wild house mice in acontrolled laboratory setting in an attempt to understand themechanisms maintaining natural MHC diversity. Three rearinggroups of wild test mice were produced: nonfostered controlmice, mice fostered into families of an inbred laboratory mousestrain, and mice fostered into families of a second mouse straindiffering genetically from the first only within the MHC region.At maturity, test mice were given a choice of two opposite-sexstimulus mice of the two MHC-congenic strains used for fostering.Test mice were scored for several measures of preference includingamount of time spent with either stimulus mouse, and ejaculationwith a stimulus mouse. Females in two of three rearing groupsspent more time with one MHC type regardless of rearing environment,suggesting that females did not prefer mates dissimilar fromfamily MHC type. Time preferences tended to be stronger in femalesthan in males. Male test mice ejaculated indiscriminantly. Femalewild mice mated to ejaculation more often in longer trials,but these matings were still too infrequent to assess preferences.Fostering had little or no effect on MHC-based mate preferencesof wild house mice, and no evidence suggested that MHC was usedto avoid inbreeding. Wild female mice may still choose matesbased on MHC haplotypes (but do not necessarily prefer MHC-dissimilarmates); other cues are probably also used. Based on these results,inbreeding avoidance does not seem a strong mechanism for maintainingnatural MHC diversity