Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-tyrosine from Hemolymph of Fleshfly Neobellieria bullata

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β‑Ala‑Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β‑Ala‑Tyr and its admixtures have been converted into preparative purification of β‑Ala‑Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β‑Ala‑Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Derivative of Scorpion Neurotoxin BeM9 Is Selective for Insect Voltage-Gated Sodium Channels

Scorpion α-toxins are small proteins inhibiting the inactivation of voltage-gated sodium channels. They can selectively act on either mammalian (mammal toxins) or insect channels (insect toxins), or affect both types of channels (α-like toxins). Currently no model has been proposed that fully explains the dependence of selectivity upon amino acid sequence, but some patterns have already been established. Thus, most mammal toxins have an aspartic acid residue in position 8, which is involved in the formation of the nest motif, but it is still not clear whether this residue interacts directly with channels. The objective of our study was to obtain a derivative of the α-like toxin BeM9 with the replacement of lysine in position 8 by glutamate (K8E), changing the charge, but excluding the formation of the nest motif. In addition, we replaced the tyrosine in position 17 with glycine (Y17G), which is characteristic of mammal toxins. Surprisingly, the double-mutant derivative BeM9EG lost its activity on mammalian channels, becoming an insect toxin. To explain these changes, we constructed models of BeM9 and BeM9EG complexes with channels, and also performed molecular dynamics of isolated toxins. Analysis of intermolecular contacts in the complexes did not explain the reason for the selectivity change. Nevertheless, the structure of intramolecular contacts and data on molecular mobility indicate an important role of residues K8 and Y17 in stabilizing a certain conformation of BeM9 loops. We assume that the replacement of these residues allosterically affects the efficiency of toxin binding to channels.

BEES AND WASPS VENOM TOXINS, ITS IMMUNE-ALLERGIC RESPONSES, DIAGNOSIS AND THERAPEUTICS

Present article explains insect toxins, its immune allergic, pharmaceutical and therapeutic effects. Insect venom glands generate enzymatic and non-enzymatic toxins and are inflicted by the stings. Insect’s envenomation are highly painful, inflamed and life-threatening. It causes breathing difficulties, bronchospasm, hypotension and arrhythmia, cardiopulmonary problems, and imposes allergic reactions. Wasp venom toxins generate strong T-cell responses in hypersensitivity patients and stimulate the production of IgE antibody molecules. Massive envenomations causes the death of victims due to the toxic effects of the venom toxins if clinical treatment is delayed. This article also emphasizes the role of natural and recombinant toxins for the development of highly sensitive immune-assays for diagnosis of allergen-specific tolerance, its early and delayed effects in patients to avoid fatal anaphylactic reactions. It also directs about the essentiality of immune diagnostics, vaccines and antiserum therapy in high population density regions where incidences of wasp and bee envenomations are more frequently occur. Venom immunotherapy can restore normal immunity against venom allergens and may also provide lifetime tolerance against venoms. This article highlights the major effects of insect venom allergens, its diagnosis and venom immunotherapy.

Anti-Insect Secondary Metabolites from Fungal Endophytes of Conifer Trees

Choristoneura fumiferana is the most economically-important insect pest in eastern North America. Historically, strategies to control epidemics have relied on chemical pesticides that are no longer approved for use. The presence of fungal endophytes in cool area grass species and their role in reducing the impact of herbivorous insects is well understood. Recent work has demonstrated that foliar endophytes of conifers also produce anti-insect toxins. Field and nursery studies testing trees infected with the rugulosin producing endophyte Phialocephala scopiformis reduced the growth and development of C. fumiferana. The study of foliar endophytes from a variety of conifers including: Picea mariana, P. rubens and P. glauca as well as Abies balsamea and Larix laricina for the discovery of other anti-insect toxins are discussed. These endophytes are horizontally transmitted thus they are not present in nursery seedlings. Inoculating seedlings with toxigenic endophyte strains has been demonstrated to be effective in providing the tree with tolerance to herbivorous insects.

Insect toxins from spruce endophytes

Extracts of fermentation cultures of a fungal endophyte (DAOM 221611) from spruce needles have afforded the known macrocyclic antibiotic vermiculin (1), 7α,8β,11-trihydroxydrimane (2), and eight novel 13-carbon γ-lactones, namely trans-3-methyldodec-cis-6-en-4-olide (3), trans-8-hydroxy-3-methyldodec-cis-6-en-4-olide (4), trans-8-acetoxy-3-methyldodec-cis-6-en-4-olide (5), trans-9-hydroxy-3-methyl-8-oxo-dodec-trans-6-en-4-olide (6), trans-8,9-dihydroxy-3-methyldodec-cis-6-en-4-olide (7), trans-9-hydroxy-8-oxo-3-methyldodecan-4-olide (8), trans-7,9-dihydroxy-3-methyl-8-oxo-dodecan-4-olide (9), and trans-6-hydroxymethyl-3-methyl-7-oxo-undecan-4-olide (10). A known JH III metabolism product, 10,11-dihydroxyfarnesenic acid (11), was also isolated and synthesized from farnesol. Other endophyte cultures from black spruce and white spruce afforded the novel 6,7-dihydroxy-2-propyl-2,4-octadien-4-olide (16), 5,6,8-trihydroxy-4-(1'-hydroxyethyl) isocoumarin (17) plus the known sescandelin (18), sescandelin B (19), and 4-hydroxy-2-methoxyacetanilide (20). Several of the γ-lactones showed toxicity to spruce budworm (Choristoneura fumiferana Clem.) larvae and vermiculin 1 and compound 16 were toxic to spruce budworm cells.Key words: toxigenic endophytes, insect toxins, γ-lactones, isocoumarins.

Interacting Domains of Anti-Insect Scorpion Toxins and their Sodium Channel Binding Sites: Structure, Cooperative Interactions with Agrochemicals, and Application

Integrated pest management in modern crop protection may combine chemical and biological insecticides, particularly due to the risks to the environment and livestock arising from the massive use of non-selective chemicals. Thus, there is a need for safer alternatives, which target insects more specifically. Scorpions produce anti-insect selective polypeptide toxins that are biodegradable and non-toxic to warm-blooded animals. Therefore, integration of these substances into insect pest control strategies is of major importance. Moreover, clarification of the molecular basis of this selectivity may provide valuable information pertinent to their receptor sites and to the future design of peptidomimetic anti-insect specific substances. These toxins may also be important for reducing the current overuse of chemical insecticides if they produce a synergistic effect with conventional pesticides. Based on these considerations, our major objectives were: 1) To elucidate the three-dimensional structure and toxic-site of scorpion excitatory, "depressant, and anti-insect alpha toxins. 2) To obtain an initial view to the sodium channel recognition sites of the above toxins by generating peptide decoys through a phage display system. 3) To investigate the synergism between toxins and chemical insecticides. Our approach was to develop a suitable expression system for toxin production in a recombinant form and for elucidation of toxin bioactive sites via mutagenesis. In parallel, the mode of action and synergistic effects of scorpion insecticidal toxins with pyrethroids were studied at the sodium channel level using electrophysiological methods. Objective 1 was achieved for the alpha toxin, LqhaIT Zilberberg et al., 1996, 1997; Tugarinov et al., 1997; Froy et al., 2002), and the excitatory toxin, Bj-xtrIT (Oren et al., 1998; Froy et al., 1999; unpublished data). The bioactive surface of the depressant toxin, LqhIT2, has been clarified and a crystal of the toxin is now being analyzed (unpublished). Objective 2 was not successful thus far as no phages that recognize the toxins were obtained. We therefore initiated recently an alternative approach, which is introduction of mutations into recombinant channels and creation of channel chimeras. Objective 3 was undertaken at Riverside and the results demonstrated synergism between LqhaIT or AaIT and pyrethroids (Lee et al., 2002). Furthermore, negative cross-resistance between pyrethroids and scorpion toxins (LqhaIT and AaIT) was demonstrated at the molecular level. Although our study did not yield a product, it paves the way for future design of selective pesticides by capitalizing on the natural competence of scorpion toxins to distinguish between sodium channels of insects and vertebrates. We also show that future application of anti-insect toxins may enable to decrease the amounts of chemical pesticides due to their synergism.