Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities.

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect.

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.

Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity.

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.

Effects of TMB-8, a putative calcium antagonist, on neuromuscular transmission and muscle contractility in the mouse phrenic nerve-hemidiaphragm preparation.

The effects of TMB-8 [8-(N.N.-diethylamino)octyl-3,4,5-trimethoxybenzoate], a putative calcium antagonist, on directly and indirectly evoked isometric twitches, tetanic contractions and potassium- and caffeine-induced contractures, were investigated in the mouse isolated phrenic nerve-hemidiaphragm preparation. In the lowest concentration tested (10(-6) M), TMB-8 produced an augmentation of both directly and indirectly induced twitches. In higher concentrations (10(-5)-3 x 10(-5) M), this augmentation was followed by twitch reduction. In the highest concentrations (10(-4) M-3 x 10(-4) M), only twitch reduction in a concentration-dependent manner was observed. TMB-8 also depressed both directly and indirectly induced tetanic contractions. However, the drug was more effective in depressing neurotransmission than in reducing muscle contractility. Elevated Ca2+ (4-8 mM) or 3,4-diaminopyridine (10(-4) M) produced a good reversal of neuromuscular blockade but this effect was transient. Pretreatment with 4 mM Ca2+ had no significant effect on the time required to produce a 50% or a 90% inhibition of directly or indirectly induced twitches. However, 8 mM Ca2+ significantly prolonged the inhibitory effects of TMB-8 on indirectly, but not directly induced twitches. On the other hand, neostigmine (3 microM) appeared to hasten the blockade of transmission. Submaximal potassium-induced contractures were markedly depressed while caffeine-induced contractures were only slightly depressed by TMB-8 in the concentration range tested (10(-5)-3 x 10(-4) M). The results are consistent with the hypothesis that TMB-8 inhibits skeletal muscle contractility by a reduction in transmembrane Ca2+ movement, a depression of postsynaptic acetylcholine receptor sensitivity, and a decreased mobilization of sequestered calcium from the sarcoplasmic reticulum.