improved in vitro efficacy was observed withit inhibitor combination

When methionine sulfoximine, glutamine synthetase inhibitor, was added to the ammonisolution or blood meal, the concentration of glutamine in hemolymph decreased significantly, whereas the ARN-509 concentration of proline increased dramatically. In the presence of azaserine, glutamate synthase inhibitor, the glutamine concentration increased whereas the proline concentration decreased significantly. This confirms the presence of glutamate synthase in mosquitoes, and suggests that the enzyme contributes to the production of glutamate for the synthesis of proline. Several key enzymes related to ammonimetabolism showed activity in homogenates of mosquito fat body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamate synthase, glutamine synthetase, pyrroline 5 carboxylate synthetase, and pyrroline 5 carboxylate reductase were cloned and sequenced. The mRNexpression patterns of these genes were examined by real time reverse transcriptase polymerase chain reaction Inguinal canal in fat body and midgut before and after blood meal. The results show that female mosquitoes have evolved efficient mechanisms to detoxify large load of ammonia. We have recently demonstrated that Aedes aegypti females are able to detoxify ammonimainly through the synthesis of glutamine and proline along with the ammonia, uric acid and ureexcretion. Now, we have established protocol to study the kinetics of incorporation of 15 N from labeled ammoniinto glutamine, glutamic acid, alanine and proline in Ae. aegypti. Mosquitoes were fed 3% sucrose solutions containing either 80 mM 15 NH4Cl or 80 mM glutamine LDN-57444 labeled with 15N in either the amide nitrogen or in both amide and amine nitrogens. In some experiments, specific inhibitors of glutamine synthetase or glutamate synthase were added to the feeding solutions. At different times post feeding which varied between 0 and 96 hours, whole mosquitoes were immersed in liquid nitrogen. Whole bodies of 10 insects were homogenized in water. The suspension was centrifuged and the supernatant collected. The samples plus deuterium labeled internal standards were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. The quantification of 15N labeled and unlabeled amino acids was performed at series of different neutral losses by carrying out multiple reaction monitoring scans in triple quadrupole mass spectrometer. The results showed that the rate of incorporation of 15N from labeled ammoniinto amino acids was rapid and that the label first appeared in the amide side chain of Gln and then in the amino group of Gln. The addition of inhibitors of key enzymes in the ammonimetabolism pathway confirmed that mosquitoes efficiently metabolize ammonithrough metabolic route that mainly involves glutamine synthetase and glutamate synthase. Moreover, complete deduced amino acid sequence for GltS of Ae. aegypti was determined. The molecular signatures involved in electron donors and the previous biochemical studies confirm that Ae.