The effect of some Solanum steroidal alkaloids and glycoalkaloids on larvae of the red flour beetle, Tribolium castaneum, and the tobacco hornworm, Manduca sexta.

Evaluation of the inhibitory effect of a series of secondary plant compounds including steroidal alkaloids and glycoalkaloids on larvae of the red flour beetle, Tribolium castaneum, was investigated. Larval growth was inhibited on artificial diets containing 1 mumol g-1 diet of the glycoalkaloids solamargine, solasonine and tomatine, whereas the corresponding aglycones solasodine and tomatidine, and also tomatidenol, were inactive. The inhibitory effect of solamargine and tomatine, but not of solasonine, was completely abolished by addition of 1 mumol g-1 diet cholesterol and/or sitosterol. Nonetheless, synthetic cholesteryl tomatide displayed significant activity at 2 mumol g-1 diet. Parallel studies with the tobacco hornworm, Manduca sexta, showed marked inhibitory activity of tomatine at a dietary concentration of 1 mumol g-1, whereas the other compounds did not affect sterol metabolism or larval development. An appraisal of the factors influencing the mode of action of the active steroidal glycoalkaloids is attempted.

Effect of insect-growth-disrupting amines and amides on Schistosoma mansoni in vitro.

A series of nonsteroidal alkylamines and alkylamides, which apparently inhibit phytosterol dealkylation in insects and free-living nematodes, as well as steps in the ecdysteroid biosynthetic pathway of insects, are also known to affect growth and development of animal-parasitic nematodes. The effect of these compounds on the motility, pairing, and viability of the trematode, Schistosoma mansoni, maintained in vitro was examined by incorporating the potential inhibitors into the medium employed for parasite culture. The alkylamines were more active than their amide counterparts, killing all parasites within 18 hr when tested at 25 parts per million. At lower concentrations, reductions in schistosome pairing and motility were observed. The functional group of the amine compounds influenced the schistosomicidal activity, with N-ethylamines being more effective than N,N-dimethylamines of identical carbon chain-length. Of all the compounds tested, N-ethyldodecanamine appeared to be the most promising schistosomicidal agent, killing all parasites within 22 hr at 10 parts per million and preventing parasite pairing at 3 parts per million.

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

Ecdysteroid production in Drosophila melanogaster reared on defined diets.

Larvae of Drosophila melanogaster were reared aseptically on defined diets containing either cholesterol, campesterol or sitosterol as the only dietary sterol. Sterol analyses of pupae revealed that insects reared on campesterol and sitosterol diets contained 3.3 and 8.1% cholesterol, indicative of an ability to accumulate this sterol. Ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids in insects from all diet studies, though makisterone A was detected in pupae reared on campesterol and sitosterol.

Sterol metabolism in the tobacco hornworm, Manduca sexta--a review.

A number of intermediates involved in the dealkylation and conversion of the major C28 and C29 phytosterols to cholesterol in insects were first isolated and identified in studies with the tobacco hornworm, Manduca sexta, carried out in our laboratory. We also investigated the effects of a variety of known sterol metabolism inhibitors in Manduca, particularly those affecting the delta 24-sterol reductase enzyme, and synthesized and tested a number of new inhibitors as well. In-depth studies of ecdysteroids in Manduca during embryogenesis and during pupal-adult development provided new information on molting hormone content, biosynthesis, and metabolism. In addition, this insect has been utilized in the study of three specific enzyme systems of ecdysteroid metabolism, namely 20-monooxygenase, 3-epimerase, and phosphotransferase, which are critical to activation and deactivation of molting hormones in insects.

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols in Manduca sexta.

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids in Manduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid alpha-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol in Manduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The delta 24-bonds of lanosterol and cycloartenol were effectively reduced by the Manduca delta 24-sterol reductase enzyme, as is the delta 24-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C28 and C29 phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C28 and C29 sterols was readily converted to cholesterol, and a significant amount of 7-dehydrocholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.

Effects of potential inhibitors on Brugia pahangi in vitro: macrofilaricidal action and inhibition of microfilarial production.

A series of compounds that apparently disrupt hormonally regulated processes in insects have been examined for effects on the viability and microfilarial production of adult Brugia pahangi cultured in vitro. The azasteroids, 25-azacoprostane and 25-azacholestane, inhibited the production of microfilariae at 5 ppm, the former also exhibiting macrofilaricidal activity at this concentration. The brassinosteroids examined inhibited microfilarial production at 5 ppm but did not affect worm viability. Azadirachtin also proved to be a significant inhibitor of microfilarial release without effect on worm motility or viability. Of all the compounds tested, the non-steroidal amines appeared to be the most promising as potential filaricides, several of them proving to be macrofilaricidal at 1 ppm and affecting microfilarial production at even lower concentrations.

Intermediates of stigmasterol metabolism in Spodoptera littoralis.

Stigmasterol-24,28-epoxide, 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol, and 22E-cholesta-5,22,24-trien-3 beta-ol were identified as normal metabolites of [3H]stigmasterol in Spodoptera littoralis larvae. Relative concentrations of all three of these metabolites increased when a diazasterol inhibitor was fed in combination with stigmasterol in the artificial diet. Identification of these sterols as intermediates in the conversion of stigmasterol to cholesterol in this insect indicates that intermediates analogous to fucosterol and fucosterol-24,28-epoxide in the conversion of sitosterol to cholesterol are produced in the metabolism of stigmasterol. This is the first published identification of stigmasterol-24,28-epoxide and 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol as intermediates in this pathway in an insect.

Metabolism of [14C]cholesterol to C-20 isomeric [14C]pregn-5-ene-3,20-diols in the tobacco hornworm, Manduca sexta.

After injection into male and female fifth-instar larvae of Manduca sexta, [14C]cholesterol was converted to C21 steroids, [14C]pregn-5-ene-3 beta,20-diols. These metabolites were isolated from 8-day-old pupae and were identified by TLC, HPLC, and GC-MS as the C-20 isomers of pregnene-3 beta,20-diol. They also were isolated from male and female meconium fluid (of 16-day-old pupae) following injection of [14C]cholesterol into 14-day-old pupae.

Use of temporary intraluminal shunts in selected peripheral arterial injuries.

Successful limb salvage following major peripheral arterial injury is now limited mainly by destruction of nerve, bone, and soft tissue. In some patients prolonged tissue ischemia may be a problem because of delays in treatment caused by associated injuries. In order to minimize tissue ischemia times, temporary arterial shunting was used in selected patients with injuries requiring fracture fixation or extensive debridement. Forty-two patients with major peripheral arterial injuries were seen in 27 months. Thirteen patients, including ten of the 12 with popliteal artery injuries, had placement of a temporary arterial shunt. All shunted patients had successful revascularization and no complications of the shunting occurred. In the total group of 42 there was one death and one below-knee amputation. In patients with extensive but salvageable injuries occurring with peripheral arterial injuries we feel that the use of a temporary arterial shunt is safe and may prevent prolonged severe ischemia.

The fate of radiolabeled steroids in ovaries and eggs of the tobacco hornworm,Manduca sexta.

To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco hornworm,Manduca sexta. Following injection of [(14)C]cholesterol, 22,25[(14)C]dideoxyecdysome or [(3)H]ecdysone into female pupae (day 16), only [(14)C]cholesterol was incorporated and metabolized. It was converted to labeled nonecdysteroid and ecdysteroid conjugates, of which the latter in ovaries and 48- to 64-hr-old eggs is mainly 26-hydroxyecdysone 26-phosphate (>85% in ovaries). Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography (RP-HPLC) showed that the levels of 26-hydroxyecdysone 26-phosphate were 31 µg/g of ovaries from 4-day-old females and 25 µg/g and 17 µg/g of 48- to 64-hr-old and 72- to 88-hr-old eggs, respectively. The RP-HPLC of the conjugate fraction of 48- to 64-hr-old eggs showed an additional peak of radioactive material eluting about three min before the 26-hydroxyecdysone 26-phosphate. The quantity of this material increased in the 72-to 88-hr-old eggs, though it was not detected in the analyses of the conjugate fraction from ovaries. Additional peaks of radioactive material eluting before the 26-hydroxyecdysone 26-phosphate peak were observed in the chromatogram of the conjugates of 72- to 88-hr-old eggs. These radioactive materials need to be identified to determine the ultimate fate of ecdysteroids in the developing embryos of the tobacco hornworm. No radioactive free ecdysteroids were detected in either egg age group.

Selective sterol transfer in the honey bee: Its significance and relationship to other hymenoptera.

The honey bee,Apis mellifera, is one of only a few species of phytophagous insects known to be unable to convert C-24 alkyl phytosterols to cholesterol. Regardless of the dietary sterols available to worker bees, the major tissue sterol of brood reared by the workers is always 24-methylenecholesterol, followed by sitosterol and isofucosterol. Normally, little or no cholesterol is present in honey bee sterols. The maintenance of high levels of certain sterols is accomplished through a selective transfer of sterols from the endogenous sterol pools of the workers to the developing larvae through the brood food material secreted from the hypopharyngeal and mandibular glands and/or the honey stomach of the workers. The selective uptake and transfer of radiolabeled C27, C28 and C29 sterols have been studied to correlate these aspects of sterol utilization with the discovery of an unusual molting hormone (ecdysteroid) in honey bee pupae as the major ecdysteroid of this stage of development. The phylogenetic implications of this selective transfer phenomenon in the honey bee and comparison with sterol metabolism in certain other hymenopteran species emphasize the diversity of steroid biochemistry in insects.

Biosynthesis of a C21 steroid conjugate in an insect. The conversion of [14C]cholesterol to 5-[14C]pregnen-3 beta,20 beta-diol glucoside in the tobacco hornworm, Manduca sexta.

Following injection into Manduca sexta (L.) female pupae (day 16), [14C]cholesterol was converted to a C21 steroid conjugate, 5-[14C]pregnen-3 beta,20 beta-diol glucoside. The conjugate was isolated from ovaries and eggs and contained three glucose units at least one of which is attached to C-20. The distribution of the other two glucose units remains to be determined. Other than the dealkylation of C-24 alkane or alkene substituents, side-chain cleavage of sterols is uncommon to insects. Here we report the first definitive proof of the biosynthesis of a C21 steroid conjugate from cholesterol in an insect species. The capability of M. sexta to so readily convert cholesterol to a C21 steroid suggests a physiological role for 5-pregnen-3 beta,20 beta-diol in this species.

Inhibition of C28 and C29 phytosterol metabolism by N,N-dimethyldodecanamine in the nematode Caenorhabditis elegans.

Effects on the metabolism of campesterol and stigmasterol in Caenorhabditis elegans were investigated using N,N-dimethyldodecanamine, a known inhibitor of growth, reproduction and the delta 24-sterol reductase of this nematode. 7-Dehydrocholesterol was the predominant sterol (51%) of C. elegans grown in stigmasterol-supplemented media, whereas addition of 25 ppm amine resulted in a large decrease in the relative percentage of 7-dehydrocholesterol (23%) and the accumulation of a substantial proportion (33%) of delta 24-sterols (e.g., cholesta-5,7,24-trienol) and delta 22,24-sterols (e.g., cholesta-5,7,22, 24-tetraenol) but yielded no delta 22-sterols. Dealkylation of stigmasterol by C. elegans proceeded in the presence of the delta 22-bond; reduction of the delta 22-bond occurred prior to delta 24-reduction. Addition of 25 ppm amine to campesterol-supplemented media altered the sterol composition of C. elegans by increasing the percentage of unmetabolized dietary campesterol from 39 to 60%, decreasing the percentage of 7-dehydrocholesterol from 26 to 12%, and causing the accumulation of several delta 24-sterols (6%). C. elegans also was shown to be capable of dealkylating a delta 24 (28)-sterol as it converted 24-methylenecholesterol to mostly 7-dehydrocholesterol. The proposed role of 24-methylenecholesterol as an intermediate between campesterol and 7-dehydrocholesterol was supported by the results.

Ecdysteroids in developing ovaries and eggs of the tobacco hornworm.

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.

Comparative effects of growth inhibitors on sterol metabolism in the nematode Caenorhabditis elegans.

An analogous series of dimethylalkyl compounds, consisting of four amines, an amide, and a phosphonate ester, inhibited motility and reproduction of the nematode Caenorhabditis elegans. Dimethylamines with straight-chain lengths of 12, 14, or 16 carbon atoms were equally active nematicides, causing greater than 80% population growth inhibition at a concentration of 25 ppm. The C12 straight-chain amine and its corresponding amide produced similar inhibition and were much more potent than either the corresponding C12 phosphonate or a C12 branched-chain amine. Inhibition of the delta 24-sterol reductase system was exhibited by all four amines, but not by the amide or phosphonate, in the following order of activity: C12 branched-chain amine greater than C12 straight-chain amine greater than C14 amine greater than C16 amine. The C12 branched amine also blocked the C-24(28)-dehydrogenase system in the conversion of sitosterol to fucosterol, the initial step in sitosterol dealkylation.

Sterol metabolism in the nematodeCaenorhabditis elegans.

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3ß-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3ß-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 µg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3ß-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3ß-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.