Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH’s preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.

Research has shown that broiler chickens reared on a low-protein diet have a more efficient protein digestion. However, information on the fate of absorbed amino acids in relation to the dietary crude protein level in poultry is sparse. Therefore, this study aimed at developing a methodology for a 1-(13)C(1)-leucine breath test combined with indirect calorimetry, and to apply this technique using broiler diets known to induce differences in protein retention. From 14 days of age onwards, broiler chickens were reared on one of two isocaloric diets with substitutions between fat and protein [low-protein (LP) vs. high-protein (HP) diet: 130.4 vs. 269 g protein/kg; and 101.8 vs. 27.9 g fat/kg]. Every 4 or 5 days, three chickens per diet were placed in the respiratory cells for 48 h. The broilers were intubated with 40 mg 1-(13)C(1)-leucine/kg body weight, followed by breath sampling for 4 h at 15-min intervals and mass spectrometric analysis of the (13)C:(12)C ratio in the samples. The CO(2) level in the respiratory cell air was monitored and excreta samples were collected. The methodology to study l[1-(13)C(1)]leucine decarboxyation in chickens using a breath test combined with indirect calorimetry was accomplished. Results of the nitrogen balance test indicated that the LP broilers had an improved dietary protein retention compared with the HP animals. Moreover, LP chickens decarboxylated a significantly lower percentage of l[1-(13)C(1)]leucine, demonstrating several 'protein- or amino acid-sparing' mechanisms in animals reared on a diet with lower protein level, both at the digestive and at the postabsorptive level.