PubMed Health⌕ Search

Biomedical subjects

L L Murdock

Publications and source records attributed to L L Murdock.

At least 19 recordsLinked to original sources
PubMed2005

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests.

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.

Animals↗
PubMed2003

Transcriptome analysis of the cowpea weevil bruchid: identification of putative proteinases and alpha-amylases associated with food breakdown.

We describe here the first systematic work to discover insect genes involved in food breakdown using a cDNA library enriched for gut-expressed transcripts from Callosobruchus maculatus. A total of 1056 clones were screened for cDNA insert-containing plasmids, and 503 nonredundant open reading frames were discovered. Twenty-three inferred genes potentially involved in digestive processes in cowpea weevil were identified, including proteinases and amylases. The predicted catalytic sites were identified in the inferred cysteine and aspartic acid proteinases, and in alpha-amylases. Transcriptome analysis of the cowpea bruchid will potentially permit gene discovery in other beetles, an insect order of major economic and ecological importance that is poorly represented in genomic databases.

Amino Acid Sequence↗
PubMed2001

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Base Sequence↗
PubMed2000

Deterministic modeling of negative cross-resistance strategies for use in transgenic host-plant resistance.

Negative cross-resistance refers to a situation in which an insect population that is tolerant (resistant, virulent) to one insecticide is hyper-sensitive (avirulent) to a second insecticide and insects hyper-sensitive to the first compound are tolerant to the second. Most research dealing with negative cross-resistance has focused on the molecular biology and chemical aspects of this phenomenon. We explored, from a population genetics perspective, whether negative cross-resistance is feasible in the control of an insect population. As a first step towards this goal, we used a deterministic approach to evaluate different control scenarios and to identify some of the potential limitations of negative cross-resistance strategies. Specifically, we investigated how such approaches could be used in a host-plant resistance program. Homo- and heterozygous insect fitness influenced the effectiveness of the toxins in controlling the insect population. The negative cross-resistance strategy was most useful when the insects' virulence to both host-plant toxins was recessive. When virulence was dominant, there were many periods when intervention with an outside (or third) class of compounds, which had a different mode of action than that of the negative cross-resistance compounds, was needed to control the insect population. The greater the number of insect generations per plant generation, in the absence of immigration or emigration in the insect population, the greater the requirement for intervention with a third class of compound to maintain effective control of the insect population. When the toxins were rotated every insect generation, and virulence in the insect was recessive to both toxins, effective control of the insect population was maintained without intervention of a third class of compounds.

Animals↗
PubMed2000

A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Amino Acid Sequence↗
PubMed1998

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II.

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

Binding Sites↗
PubMed1998

Phage display selection can differentiate insecticidal activity of soybean cystatins.

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200-1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.

Amino Acid Sequence↗
PubMed1996

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II.

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp88, Glu108, Tyr134, Asn136, Leu226 and Gln227) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at Asp88, Tyr134 or Asn136 eliminated GlcNAc binding activity by GS-II, while those at Glu108, Leu226 or Gln227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu226 and Gln227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.

Acetylglucosamine↗
PubMed1996

An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae).

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

Acetylglucosamine↗
PubMed1992

Central projections of axons from taste hairs on the labellum and tarsi of the blowfly, Phormia regina Meigen.

Taste hairs are located on the labellum and tarsi of blowflies. These multimodal hairs consist of four functionally distinct chemoreceptors and a mechanoreceptor. By staining selected multimodal hairs, we sought to identify the central projection patterns of multiple and single axons from those hairs. On each side of the labellum there are 11 "largest" hairs (LH). The neurons associated with the anteriormost (LH-1), posteriormost (LH-11), and one lateral (LH-6) hair on the labellum were stained selectively with cobaltous sulfide. The overall projection pattern in the central nervous system (CNS) for axons from LH-1 and LH-11 is similar and differs markedly from axons from LH-6. At least three individual axon-projection patterns were determined for each labellar hair filled, indicating a partial functional organization for axons from multimodal hairs. One identified axon, the dorsalmost axon, has terminal arborizations that do not differ with the location of its associated hair. Another axon, thicker than the others, projects to a region that is distinct from the four thin axons. Within this region the arborizations of the thick axons occupy different areas depending on the location of their associated hair. Neurons from the largest hairs on the distalmost tarsomere (D5) of each leg were also stained and consisted of one thick and four thin axons. All axons except one thin axon from tarsal D5 hairs terminate in their respective leg neuromeres. The remaining thin axon projects to the suboesophageal ganglion ipsilateral to the hair filled and terminates in the same region as a branch of the labellar dorsalmost axon. These data suggest that axonal arbors from multimodal hairs have a limited functional and somatotopic organization in the blowfly CNS.

Animals↗
PubMed1991

Alpha-amylase inhibitor, not phytohemagglutinin, explains resistance of common bean seeds to cowpea weevil.

There are claims that phytohemagglutinin (PHA), the lectin of common bean, Phaseolus vulgaris, is toxic when fed to the cowpea weevil, Callosobruchus maculatus, and that PHA serves as the chemical defense against this seed-feeding bruchid beetle (DH Janzen, HB Juster, IE Liener [1976] Science 192: 795-796; AMR Gatehouse, FM Dewey, J Dove, KA Fenton, A Pusztai [1984] J Sci Food Agric 35: 373-380). However, our studies indicate that neither PHA nor its isolectins have detrimental effects when fed to the cowpea weevil. To explain these contradictory results we characterized the commercial lectin source used by A. M. R. Gatehouse, F. M. Dewey, J. Dove, K. A. Fenton, A. Pusztai (1984, J Sci Food Agric 35: 373-380). We demonstrate here that the toxic effects of PHA to cowpea weevil are due to an alpha-amylase inhibitor contaminant in the commercial preparation.

Journal Article↗
PubMed1990

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development.

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety "Red Kidney."

Journal Article↗
PubMed1987

Amphetamine and reserpine deplete brain biogenic amines and alter blow fly feeding behavior.

HPLC with electrochemical detection was used to determine the levels of p-hydroxyphenylethanolamine (octopamine), 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (5-HT) in the brains of control, reserpine, and d-amphetamine-treated blow flies, Phormia regina Meigen. Parallel studies were carried out to assess the effects of the two drugs on fly feeding behavior, measured as mean acceptance threshold: the minimum sucrose concentration to which the average fly in a population will respond by proboscis extension when its tarsi contact the solution. In saline-injected control flies, all three amines were found at levels of approximately 2 pmol/brain. Thirty minutes after injection with d-amphetamine (12 micrograms/fly), brain octopamine was depleted by 85%, whereas dopamine and 5-HT were depleted by 70%. Reserpine (5 micrograms/fly) caused 70% depletion of dopamine and greater than 90% depletion of both octopamine and 5-HT 24 h after injection. However, the effect of reserpine was much slower in onset (hours versus minutes) and more persistent (days versus hours) than was the effect of d-amphetamine. With either drug, the time course of amine depletion closely matched the time course of the increase in feeding threshold observed in drug-treated flies. These results suggest that CNS pools of the biogenic amines, octopamine, dopamine, and 5-HT are important in governing blow fly responsiveness to food stimuli.

Amines↗
PubMed1986

Effects of substituted phenylethylamines on blowfly feeding behavior.

The pharmacology of adult Phormia regina (Meigen) feeding behavior was explored by injecting candidate drugs into starved blowflies and then determining their responsiveness to aqueous sucrose, via the proboscis extension reflex. d-Amphetamine caused responsiveness to fall dramatically, while related drugs and biogenic amines had varying effects. When d-amphetamine-treated flies were fed 1 M sucrose, they consumed significantly more than control flies. Electrophysiological studies demonstrated that the responses of tarsal sugar receptor neurons to aqueous sucrose were not significantly altered by d-amphetamine. These observations are compatible with our hypothesis that octopamine positively modulates blowfly feeding behavior and suggest that other aromatic biogenic amines affect feeding behavior in this insect.

Animals↗
PubMed1983

Stimulation of blowfly feeding behavior by octopaminergic drugs.

Adult blowflies (Phormia regina Meigen) injected with the octopaminergic drug demethylchlordimeform (10 mug per fly) exhibited enhanced proboscis extension responses when their tarsae were touched to water or aqueous sucrose. They drank more water than saline-injected control flies did but the quantity imbibed was within the normal fluid intake capacity. They became grossly hyperphagic when offered 1 M sucrose, doubling (and in some cases even tripling) their initial body weights. Three other drugs enhanced tarsal responsiveness and induced hyperphagia: DL-octopamine, clonidine (which is known to stimulate octopaminergic receptors in insects), and pargyline, a monoamine oxidase inhibitor. Yohimbine, an antagonist of one class of octopaminergic receptor in insects, prevented the hyperphagia induced by all four drugs. Dopamine, 5-hydroxytryptamine, and DL-norepinephrine failed to cause hyperphagia. These results suggest that octopaminergic receptors in the nervous system of the blowfly positively modulate feeding and drinking behavior.

Journal Article↗