Single-pulse magneto-optic microscopy: a new tool for studying optically induced magnetization reversals.

We have developed a femtosecond magneto-optical imaging system that allows measurements of permanent magnetic effects that are initiated by a single excitation pulse. The system combines a subpicosecond temporal resolution and a high spatial resolution. We demonstrate the system in an experiment that studies the laser-induced magnetization reversal in ferromagnetic thin films.

Porin isolated from the outer membrane of Erwinia amylovora and its encoding gene.

A major Erwinia amylovora outer-membrane protein (Omp-EA) and the gene encoding for this protein (omp-EA) were isolated and characterized. The native Omp-EA protein forms a trimeric structure of approximately 114 kDa. This protein demonstrated high resistance to detergents such as SDS and octyl-glucopyranoside, but disaggregated to monomers with a molecular weight (MW) of approximately 39 kDa after heating at 95 degrees C for 10 minutes in sample buffer. The pore-forming ability of the oligomeric Omp-EA was determined by the liposome swelling assay, demonstrating that the oligomeric protein formed nonspecific channels with an exclusion limit of approximately 660 Da. On dissociation, the monomers did not exhibit pore-forming ability. The omp-EA gene was cloned and sequenced (GenBank Accession No. DQ184680). Sequence analysis revealed an open reading frame of 1152 bases. The deduced amino-acid sequence had 383 amino acids. The mature protein consisted of 362 amino acids and had a calculated MW of 39,210 Da. Multiple-sequence alignment of Omp-EA with other porins from the Enterobacteriaceae family revealed 51% to 63% identity. The first 16 amino acids from the N-terminal exhibited the highest identity (100%) to the porins OmpC, OmpF, and PhoE of Escherichia coli. Two methods were used to predict the secondary structure: APSSP2 and Hidden and Markov's model. The monomers of Omp-EA porin presented a topology of 16 transmembranal beta-strands. The area of the loops between the beta -strands was proposed. It is suggested that further research on the porin and its loops may be important for understanding the mechanism of E. amylovor to invade plant tissues.

Targeting of an expressed neurotoxin by its recombinant baculovirus.

AaIT, an insect-selective neurotoxic polypeptide derived from scorpion venom, has recently been used to engineer recombinant baculoviruses for insect pest control. Lepidopterous larvae infected with an AaIT-expressing baculovirus reveal symptoms of paralysis identical to those induced by injection of the native toxin. However, the paralyzed larvae treated by the recombinant virus possess an approximately 50-fold lower hemolymph toxin concentration than insects paralyzed by the native toxin. The mechanism of this potentiation effect was studied using immunocytochemistry, electrophysiology and toxicity assays. (i) Light microscopy, using peroxidase-conjugated antibodies, revealed the presence of toxin in virus-susceptible tissues, including tracheal epithelia located close to the central nervous system and beyond its lamellar enveloping sheath. (ii) High-resolution immunogold electron microscopical cytochemistry clearly revealed the presence of recombinant AaIT toxin inside the thoracic and abdominal ganglia on neuronal cell bodies and axonal membranes. (iii) Ventral nerve cords dissected from silkworm larvae infected with the recombinant baculovirus exhibited a high degree of excitability, expressed as enhanced frequency and bursting mode of their spontaneous activity, when compared to nerve cords infected with the wild-type virus. We conclude that the recombinant-virus-infected tracheal epithelia, outbranching in the body of an infected insect, (i) locally supply a continuous, freshly produced toxin to its neuronal receptors and (ii) introduce the expressed toxin to the insect central nervous system, thus providing it with critical target sites that are inaccessible to the native toxin.

AaIT: from neurotoxin to insecticide.

AaIT is a single chain neurotoxic polypeptide derived from the venom of the Buthid scorpion Androctonus australis Hector, composed of 70 amino acids cross-linked by four disulfide bridges. Its strict selectivity for insects has been documented by toxicity, electrophysiological and ligand receptor binding assays. These last have shown that various insect neuronal membranes possess a single class of non-interacting AaIT binding sites of high affinity (K(D) = 1-3(n)M) and low capacity (0.5-2.0 pmol/mg prot.). The fast excitatory paralysis induced by AaIT is a result of a presynaptic effect, namely the induction of a repetitive firing in the terminal branches of the insect's motor nerves resulting in a massive and uncoordinated stimulation of the respective skeletal muscles. The neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loops of the insect voltage-dependent sodium channel and modification of its gating mechanism. From a strictly agrotechnical point of view AaIT involvement in plant protection has taken the following two complementary forms: firstly, as a factor for the genetic engineering of insect infective baculoviruses resulting in potent and selective bio-insecticides. The efficacy of the AaIT-expressing, recombinant baculovirus is attributed mainly to its ability to continuously provide and translocate the gene of the expressed toxin to the insect central nervous system; secondly, based on the pharmacological flexibility of the voltage-gated sodium channel, as a device for insecticide resistance management. Channel mutations conferring resistance to a given class of insecticidal agents (such as the KDR phenomenon) may greatly increase susceptibility to the AaIT expressing bioinsecticides. Thus the AaIT is a pharmacological tool for the study of insect neuronal excitability and chemical ecology and the development of new approaches to insect control.