No abstract

We investigated the mode of entry of pyrethroids into the insect body using adult housefly, Musca domestica L., as an insect model. The wings of adult female houseflies were removed, and empenthrin was applied topically to three different sites: the mesothoracic spiracle, the ventral mesothorax, and the dorsal mesothorax. Among these treatments, the application of the compound to the mesothoracic spiracle led to the quickest knockdown of the flies. To determine the importance of the spiracle as a primary entry site for the pyrethroid, knockdown times were compared between houseflies with blocked and non-blocked spiracles, using two bioassays: a vapor action test using technical grade empenthrin, and a mosquito coil test using empenthrin-impregnated coils. In both tests, the times required for 50 % knockdown of spiracle-blocked houseflies were significantly higher than those required for the non-blocked flies. However, the mortality rates of the two groups were nearly identical, suggesting that spiracles play an important role in the knockdown of houseflies. These results also suggest that the rate of pyrethroid uptake through the spiracles was decreased due to the blocking of the mesothoracic spiracle. Therefore, the spiracle may be considered the main entry site for vaporized pyrethroids.

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln 400 and Ile 404 in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.