Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA).

Background: New bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide.

Cost-effective and reliable genomic DNA extraction from plant seedlings for high-throughput genotyping in seed industries.

Genetic purity of seeds is one of the critical aspects in the seed industry. Molecular seed testing laboratories are utilizing PCR based diagnostic tools for genetic purity analysis. High quality DNA is an essential prerequisite for such analyses.

Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin).

Background: Spear®-T sold as a contact foliar spray for the control of glasshouse pests such as aphids, thrips, spider mites and whiteflies, contains the recombinant spider venom peptide GS-ω/κ-HxTx-Hv1h (named as GS-ω/κ-HxTx-Hv1a by Vestaron) as the active ingredient. Here we investigate whether fusion of the peptide to snowdrop lectin, (Galanthus nivalis agglutinin; GNA) enhances the efficacy of this venom peptide towards aphid pests.

Results: Recombinant GS-ω/κ-HxTx-Hv1h (HxTx-Hv1h) and an HxTx-Hv1h/GNA fusion protein were produced using the yeast Pichia pastoris.

Recombinant Lectin from Tepary Bean () with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification.

Herein, we report the production of a recombinant Tepary bean lectin (TBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. TBL-1 was expressed in yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that TBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca and Mn) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.

Insecticidal effects of dsRNA targeting the Diap1 gene in dipteran pests.

The Drosophila melanogaster (fruit fly) gene Diap1 encodes a protein referred to as DIAP1 (D rosophila Inhibitor of Apoptosis Protein 1) that acts to supress apoptosis in "normal" cells in the fly. In this study we investigate the use of RNA interference (RNAi) to control two dipteran pests, Musca domestica and Delia radicum, by disrupting the control of apoptosis. Larval injections of 125-500 ng of Diap1 dsRNA resulted in dose-dependent mortality which was shown to be attributable to down-regulation of target mRNA.

Sublethal effects of the insecticidal fusion protein ω-ACTX-Hv1a/GNA on the parasitoid Eulophus pennicornis via its host Lacanobia oleracea.

Background: The neurotoxin peptide ω-ACTX-Hv1a, fused to the carrier molecule GNA, presents potential for insect control as a biopesticide, being orally toxic to insect pests from different orders. However, thorough evaluation is required to assure its safety towards non-target invertebrates. Effects of this novel biopesticide on the parasitoid Eulophus pennicornis via its host Lacanobia oleracea are presented.

Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

Background: The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh.

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains.

Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris.

A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host.

Fusion to snowdrop lectin magnifies the oral activity of insecticidal ω-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system.

Background: The spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a "carrier" in order to enhance the oral activity of Hv1a.

Insecticidal activity of wheat Hessian fly responsive proteins HFR-1 and HFR-3 towards a non-target wheat pest, cereal aphid (Sitobion avenae F.).

The interaction between Hessian fly (Mayetiola destructor) and wheat (Triticum aestivum) involves a gene-for-gene resistance mechanism. The incompatible interaction leading to resistance involves up-regulation of several Hfr (Hessian fly responsive) genes encoding proteins with potential insecticidal activity. The encoded proteins HFR-1, HFR-2 and HFR-3 all possess lectin-like domains.

Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors.

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo.

Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): basis of insecticidal activity.

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.

Winged bean chymotrypsin inhibitors retard growth of Helicoverpa armigera.

Two putative Kunitz-type chymotrypsin inhibitor genes (WCI2 and WCI5) were isolated from winged bean (Psophocarpus tetragonolobus (L.) DC). While WCI2 has previously been characterized, WCI5 represents a new member of the WCI family.

Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not.

Unusual volar dislocation of the lunate into the distal forearm: case report.

A case of a severe wrist injury (fractures of the scaphoid, capitate, hamate, and ulnar styloid process) with volar dislocation of the lunate into the soft tissues of the forearm is presented. This degree of displacement of the lunate is exceedingly rare, and we believe has not been previously reported. Possible mechanisms, hyperextension injury forcing the lunate forward out of the carpus, or contact between the volar wrist skin surface and another object or surface propelling the dislocated lunate into the forearm, are discussed.

Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1.

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formed bacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteria when grown aerobically ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observed under continuous dark or light growth conditions.

Involvement of an inducible factor in interferon-gamma-mediated accumulation of HLA gene transcripts.

Earlier studies with a cDNA clone (C5-4) complementary to an interferon (IFN)-gamma-inducible mRNA showed that in human fibroblasts (FS-4), IFN-gamma induced the transcription of the cognate gene, but it required new protein synthesis (Caplen and Gupta, J. Biol. Chem.