De novo synthesis of contact female sex pheromone and hydrocarbons in Blattella germanica was examined using short in vivo incubations. Accumulation of pheromone on the epicuticular surface and the internal pheromone titer were related to age-specific changes in hydrocarbon synthesis and accumulation in normal and allatectomized females. The incorporation of radiolabel from [1-14C]propionate into the cuticular methyl ketone pheromone fraction was positively related to corpora allata activity during two gonotrophic cycles. During peak pheromone production the total internal lipid fraction contained greater titers of pheromone than the cuticular surface, and it too exhibited a cycle internally, preceding the rise in external pheromone. This suggests that synthesis and accumulation of pheromone internally are followed by transport of pheromone to the epicuticular surface where it accumulates. Radiolabel was incorporated efficiently into both cuticular and internal hydrocarbons after the imaginal molt and until the peak of pheromone synthesis, but it declined to lower levels before ovulation and throughout pregnancy. The internal hydrocarbon titer decreased 58% after oviposition, suggesting deposition in the egg case. It remained relatively unchanged during pregnancy and increased again during the second gonotrophic cycle. In allatectomized females, hydrocarbon synthesis was reduced relative to control females until oviposition in the latter. However, subsequent rates of hydrocarbon synthesis in allatectomized females (without oothecae) exceeded the rates in sham-operated females (with oothecae). In the absence of ovarian uptake of hydrocarbons, the internal titer increased without the decline found in control females at oviposition. As internal hydrocarbons increased, so did cuticular hydrocarbons and both internal and cuticular methyl ketone pheromones. These patterns corresponded well with feeding patterns in sham-operated and allatectomized females, suggesting that pheromone production is normally regulated by stage-specific feeding-induced hydrocarbon synthesis (precursor accumulation internally) and juvenile hormone-induced conversion of hydrocarbon to pheromone. They also suggest that both the cuticle and the ovaries might be target sites for hydrocarbon and possibly methyl ketone deposition.

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day8. Three factors were identified as the major contributors to this difference: I) an experimental artifact of selection for sexually receptive females, 2) a positive effect of grouping on J H synthesis and oocyte maturation, and 3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating i s low, as in experiments with isolated females that are exposed to males for a short period of time. Long-term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA-corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.

Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs). The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively. Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), we sought to define experimental conditions under which EcR-dependent transcription could be promoted by JH. Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect orthologue, USP. Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner. Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E. EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains were responsive to ecdysteroids. Again, the effects of JHIII and 20E were associated with specific partners of the chimeric EcRs. In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD. Our results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation.

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The polymerase chain reaction was used to randomly mutagenize a cDNA cassette encoding amino acids 691-946 of the sheep Na,K-ATPase ␣ subunit. The mutagenized cassettes were used to replace the wild-type region in the full-length cDNA, and pools of mutants were transfected into HeLa cells. After the generation of resistant cells via selection in 0.5 M ouabain, polymerase chain reaction was used to amplify the mutagenized cassette from the genomic DNA of the stable transfectants. Sequence analysis of the polymerase chain reaction product revealed three amino acid substitutions: I729V, L793P, and K836R. Subsequent site-directed mutagenesis experiments showed that only L793P was important for resistance. To elucidate the role of L793 in ouabain inhibition, additional mutations at this position were prepared. L793A and L793I mutants were constructed and expressed in HeLa cells. Only L793A survived selection using ouabain, which suggested that resistance is not due to the specific substitution of leucine with proline. To explore the mechanism of resistance, apparent affinities of the L793P mutant for sodium and potassium were compared to the wild-type HeLa pump. Although the apparent affinities were comparable for sodium, the mutant had a 2-fold higher apparent affinity for potassium. This suggests that the mechanism of ouabain insensitivity of L793P is due to a perturbation in the region of the enzyme that may include the K ؉ binding site.Na,K-ATPase is located on the cell membrane of all eukaryotic cells and maintains the electrochemical gradient of the cell. It consists of a larger ␣ subunit and a smaller, glycosylated ␤ subunit. The former is the catalytic subunit; it pumps three Na ϩ ions out in exchange for two K ϩ ions pumped into the cell at the expense of ATP. It is also the site of interaction with cardiac glycosides, compounds that are commonly used to treat congestive heart failure. There is considerable interest in the characterization of the ouabain binding site of Na,K-ATPase to better understand the ouabain binding site with the aim of promoting rational drug design.The site of cardiac glycoside binding is not fully understood, in part because the three-dimensional structure of the enzyme has not been elucidated. The amino-terminal half of the protein was found to be important after a chimera comprising the amino-terminal half of the ouabain-resistant rat ␣1 enzyme and the carboxyl-terminal half of the ouabain-sensitive sheep ␣1 enzyme proved to be ouabain resistant (1). Subsequently, several amino acids in the amino-terminal half of the protein were identified that affected inhibition of the enzyme by ouabain. Using the numbering of Shull et al. (2), these include the cysteine and tyrosine in the first transmembrane domain; Cys-104 (3, 4) and Tyr-108 (4); Gln-111 (1, 2), Pro-118 (4), Asp-121 (5), and Asn-122 (1, 6) in the first extracellular domain; and Tyr-308, located in the extracellular domain between the putative H3-H4 transmembrane domains (7).The possibility that the carboxyl half of the prote...