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At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, [Pro13]alpha A-conotoxin PIVA and [Pro7,13]alpha A-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with alpha-bungarotoxin demonstrate that alpha A-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

Silicates causing pneumoconiosis function as Fenton catalysts to generate hydroxyl radicals (.OH) when incubated with hydrogen peroxide and a reducing substance. In contrast, silicates which do not cause pneumoconiosis demonstrate no Fenton activity. Catalytic activity is decreased by pretreatment of silicates with the iron chelators deferoxamine or transferrin. Hemolysis from silicates is decreased by interventions which remove superoxide anion or hydrogen peroxide from the medium, or by pretreatment of dusts with iron chelators. Thus, asbestos and nonfibrous silicates may cause pneumoconiosis through a common oxidant mechanism by catalyzing production of toxic .OH radicals in the lung.