Dan E. Farnsworth

Identification of an allatostatin from adult Diploptera punctata

A peptide (allatostatin) causing strong and rapid inhibition of juvenile hormone synthesis in vitro by corpora allata from reproductively active females has been isolated from brain/retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this 13-residue peptide has been determined: Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2. Removal of the terminal amide group caused at least a ten thousandfold loss of activity. This neurohormone has no sequence similarity with any other known neuropeptide. Its target in the biosynthetic pathway is located prior to the conversion of farnesol to juvenile hormone.

Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30-40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 microM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other development stages.

Aldrin epoxidase activity and cytochrome P-450 content of microsomes prepared from alfalfa and cabbage looper larvae fed various plant diets

Abstract The alfalfa looper ( Californica autographica ) and the cabbage looper ( Trichoplusia ni ) were reared through the fourth instar on a semidefined artificial diet and for the first 60 hr of the last instar on fresh leaves of one of their host plants. Microsomes prepared from midguts and a carcass segment which included the fat body were then assayed for aldrin epoxidase activity and cytochrome P -450 content. Epoxidation by midgut microsomes of alfalfa loopers fed peppermint leaves was at a rate up to eight times that of larvae fed alfalfa, snap beans, or broccoli. The epoxidase activity of the carcass microsomes was induced about fourfold by the peppermint diet. Midgut microsomes prepared from cabbage looper larvae reared on peppermint leaves were also more active (about four times) than those reared on alfalfa, broccoli, or cabbage. Although the epoxidase activity of the carcass microsomes of the cabbage loopers was low, about one-eighth that of the midgut microsomes, this enzyme was greatly induced by peppermint leaves. In neither species did the peppermint leaf diet cause a corresponding increase in the cytochrome P -450 content of the microsomes, the maximum difference between peppermint and the other plants being about twofold. Bioassays of cabbage looper larvae reared on broccoli or peppermint and of alfalfa loopers reared on alfalfa or peppermint indicated that the stimulation of microsomal oxidase activity by the peppermint constituents provided increased tolerance for carbaryl and methomyl but not acephate.

Structure/activity studies on 1,5-disubstituted imidazoles as inhibitors of juvenile hormone biosynthesis in isolated corpora allata of the cockroach diploptera punctata

Abstract The activity of eight substituted imidazoles as inhibitors of juvenile hormone III synthesis in the cockroach, Diploptera punctata , was measured in a short-term in vitro radiochemical assay. The potency of these compounds (IC 50 of juvenile hormone III synthesis) ranged from 80 n M for KK-98 to 48 μ M for KK-71. For some compounds (KK-71, KK-51), inhibition of juvenile hormone III synthesis was not accompanied by an accumulation of the immediate precursor methyl farnesoate within the glands. For other compounds (KK-83, KK-96, KK-98) significant accumulation (i.e., more than 100 pmol/pair) of methyl farnesoate was observed. The results suggest that some substituted imidazole compounds are selective inhibitors of the corpus allatum methyl farnesoate epoxidase at low concentrations, but all inhibit methyl farnesoate synthesis at higher concentrations. Experiments with farnesol and farnesoic acid, two precursors of juvenile hormone III synthesis, suggested that the imidazole compounds may inhibit farnesoic acid O -methyl transferase in addition to their predicted inhibition of the epoxidase.

Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in susceptible and resistant strains of Musca domestica

Abstract Development and phenobarbital (PB) induction of microsomal cytochrome P -450, cytochrome P -450 reductase, two epoxidation, and two O -demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P -450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min −1 mg protein −1 ) in all enzymes returned to control values in 24 hr while metabolic capacity (product min −1 insect −1 ) achieved control values within 48 hr. The changes in turnover numbers (pmol product min −1 pmol P -450 −1 ), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate.

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12‐myristate 13‐acetate (PMA), phorbol 12,13‐dibutyrate and 1‐oleyl‐2‐acetylglycerol. 4α‐Phorbol 12,13‐didecanoate and diolein are inactive. The inhibitory effect of phorbol 12‐myristate 13‐acetate is fully reversed by 2E,6E‐farnesol or by 2E,6E‐farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20‐hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Abstract Two cytochrome P -450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P -450-1 and P -450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P -450 reductase and phospholipid. The aldrin epoxidase activity of fraction P -450-1 was twice that of P -450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O -Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P -450-2 fraction.

Tyrosinase as an inhibitor of aldrin epoxidase in house fly microsomes

Abstract The alleged inhibition by the house fly tyrosinase system of microsomal aldrin epoxidase and its reversal by KCN, which has been reported by another laboratory, was investigated. It was found that, except in newly emerged adults or in late instar larvae, no such effect was evident. Although a full explanation of the reported low epoxidase activity was not possible, some contributing practices were identified. One of these was the use of highly concentrated homogenates in the preparation of house fly microsomes, resulting in low recoveries of enzyme activity. The stimulatory effect of KCN, added during homogenization, is attributed, at least in part, to its negative effect on the determination of microsomal protein, thus resulting in a positive error when epoxidase activity is based on microsomal protein. Tyrosinase is a potent inhibitor of aldrin epoxidase in the microsomes prepared from late instar house fly larvae and its effect can be greatly reduced by the use of KCN. Bovine serum albumin, added during homogenization of house fly larvae, also enhances the epoxidase. Together, KCN and BSA, can increase epoxidase activity sevenfold in such larvae.

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in the black blow fly (Phormia regina, Meigen)

Abstract Development and phenobarbital (PB) induction of microsomal cytochrome P -450, NADPH-cytochrome c ( P -450) reductase, two epoxidation, and two O -demethylation activities were examined in chronologically timed populations of female black blow flies ( Phormia regina , Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O -demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O -demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P -450 and 5-fold for NADPH-cytochrome c ( P -450) reductase. These data suggest that PB induces several P -450s in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.