Isolation and characterization of Brush border fragments from mosquito mesenterons

Houk, Edward J.; Arcus, Yvonne M.; Hardy, James L.

doi:10.1002/arch.940030204

Cited by 29 publications

(17 citation statements)

References 33 publications

(54 reference statements)

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“…Electron microscopy of insect midgut microvilli preparations (Houk et al 1986, Santos et al 1986) demonstrates, as previously observed for vertebrate enterocytes (Schmitz et al 1973), that they are substantially free from other cell structures, although the microvilli still contain some cytoskeleton elements. The amount of contaminating membranes is evaluated with marker enzymes.…”

supporting

confidence: 59%

“…Early attempts to study the biochemistry of microvillar membranes consisted on the determination of the ratio of phosphorus to protein content in microvilli preparations from Lepidoptera larvae and SDS-PAGE of these proteins (Wolfersberger et al 1987) and in similar preparations from Diptera adults (Houk et al 1986). Nevertheless, as discussed above, microvillar membranes are contaminated by cytoskeleton elements and other minor components.…”

Section: Biochemistry Of Microvillar Membranesmentioning

confidence: 99%

“…Thus, γ -glutamyl transferase, which is a useful microvillar membrane marker for Diptera (Bodnaryk et al 1974, Espinoza-Fuentes et al 1987, Lepidoptera (Eisen et al 1989) and Dictyoptera (Parenti et al 1986), occurs only in trace amounts in Coleoptera (Ferreira et al 1990) and only in soluble form in Hymenoptera (bees, wasps, and ants) (Schumaker et al 1993). Alkaline phosphatase, a plasma membrane (microvillar or microvillar plus basolateral membranes) marker in most insects (Terra and Ferreira 1994), is a soluble enzyme in larvae of Coleoptera (Ferreira et al 1990, Reuveni et al 1993) and in adults of Diptera (Houk et al 1986) and Lepidoptera (Terra et al 1990). Acid phosphatase, which is a marker of lysosomes in some tissue (e.g.…”

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confidence: 99%

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Plasma membranes from insect midgut cells

Terra

Costa

Ferreira

2006

An. Acad. Bras. Ciênc.

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Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modifi ed into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffi c inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifi cations are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane) observed in the midgut cells of hemipterans (aphids and bugs). The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content) that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.

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supporting

confidence: 59%

Section: Biochemistry Of Microvillar Membranesmentioning

confidence: 99%

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confidence: 99%

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Plasma membranes from insect midgut cells

Terra

Costa

Ferreira

2006

An. Acad. Bras. Ciênc.

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“…The preparation was obtained from fresh or frozen whole larvae (1-3 g wet mass), stored without buffer at -80°C. The protocol was developed from previously described techniques by means of selective divalent-cation precipitations (Biber et al, 1981;Houk et al, 1986;Nielsen-LeRoux and Charles, 1992) and differential centrifugations of brush-border membranes in ice-cold 0.3 M mannitol, 5 mM EGTA, 20 mM Tris/HCl, pH 7.5. The protocol is shown schematically in Fig.…”

Section: Methodsmentioning

confidence: 99%

Binding Kinetics of Bacillus Sphaericus Binary Toxin to Midgut Brush‐Border Membranes of Anopheles and Culex sp. Mosquito Larvae

Silva-Filha

Nielsen-LeRoux

Charles

1997

European Journal of Biochemistry

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Direct-binding assays and homologous-competition assays were used to identify specific binding between the radiolabelled toxin of Bacillus sphaericus and brush-border membrane fractions (BBMF) from Anopheles gambiae and Anopheles stephensi, obtained from whole larvae preparations. In both species, the toxin bound to a single class of receptors. BBMF of A. gunzbiae had the highest binding affinity for the toxin of the species tested, with a dissociation constant ( K J of 30 2 15 nM and a maximum receptor concentration of 5 2 1 pmol/mg. Toxin binding to A. gambiae BBMF was compared with that to BBMF from B. sphaericus-susceptible (IP) and B. sphaericus-resistant (SPHAE) Culex pipiens populations. BBMF toxin binding was slower in A. gambiae than in the C. pipiens populations. The BBMF of the B. sphaericus-resistant population of C. pipiens had an association profile that was similar to the susceptible population, despite of the lack of susceptibility in vivo. No relationship between toxicity and irreversibility of toxin binding was detected. On the contrary, toxin dissociation from BBMF was fast and almost complete in BBMF of all species studied.

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“…aegypti larvae following methods modified from those of Biber et al (1981;described in Wolfersberger et al, 1987) and Houk et al (1986). Fourthinstar larvae were chilled on ice, midguts extruded by dissection under a binocular microscope, peritrophic membranes and malpighian tubules removed, and midguts rinsed in icecold buffer A (0.3 M mannitol/5 mM EGTAj20 mM Tris/ HCl, pH 7.4).…”

Section: Brush-border Membrane Isolationmentioning

confidence: 99%

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

Nielsen-LeRoux

Charles

1992

European Journal of Biochemistry

123

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The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from C u k x pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Bacillus sphaericus is a Gram-positive spore-forming bacterium and is one of the most promising agents for the control of mosquitoes. Some strains produce a very high larvicidal activity, especially for mosquito species belonging to the Culex and Anopheles genera, while they are almost inactive on Aedes species. The insecticidal activity is directly related to the synthesis of parasporal protein crystals during sporulation (Davidson, 1983;Payne and Davidson, 1984;Kalfon et al., 1984;Baumann et al., 1985). These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al., 1988;Arapinis et al., 1988; for complete review see Baumann et al., 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al., 1988;de la Torre et al., 1989;Broadwell et al., 1990). However, transformants expressing both genes are fully toxic, indicating that the protein crystal of B. sphaericus acts as a binary toxin (Broadwell et al., 1990;Baumann and Baumann, 1991).Little is known about the mode of action of these toxins, except that they appear to exert cytopathological effects (Davidson, 1981;Karch and Coz, 1983; Charles, 1987) by binding to susceptible mosquito larval midgut cells (Davidson et al., 1987;Davidson, 1988Davidson, ,1989. In addition, both proteins bind to the gastric caecae and posterior midgut of Culex larvae when administered together (Davidson et al., 1990).In this report, we demonstrate that B. sphaericus binary toxin specifically binds to susceptible mosquito larval midgut membranes, and we measure the affinity of the toxin for its putative membrane receptor. MATERIALS AND METHODS Insects and toxicity assaysLarvae of Culexpipiens ssp. pipiens L. (Montpellier strain) and Aedes aegypti L. (Bora-Bora strain) were reared on cat biscuits at 28 "C. Adult populations were maintained on honey solution, at 2 8 T , in 80% relative humidity with a 16 hi8 h photoperiod. Females were fed on guinea pigs.Toxicity assays were performed on early fourth-instar larvae, in 5-cm diameter Petri dishes containing 10 ml bacterial or crystal suspension, or soluble protein dilutions in demineralized water. Dead larvae were counted after 24 h and 48 h, and lethal concentrations killing 50% of the population (LC,,) evaluated by probit analysis (Finney, 1971). Brush-border membrane isolationBrush-border membrane fractions (BBMF) were prepared from midguts of C. pipiens and A . aegypti larvae following methods modified from those of Biber et al. (1981; described in Wolfersberger et al., 1987) and Houk et al. (1986). Fourthi...

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Isolation and characterization of Brush border fragments from mosquito mesenterons

Cited by 29 publications

References 33 publications

Plasma membranes from insect midgut cells

Plasma membranes from insect midgut cells

Binding Kinetics of Bacillus Sphaericus Binary Toxin to Midgut Brush‐Border Membranes of Anopheles and Culex sp. Mosquito Larvae

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

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