Juvenile hormone production by corpora allta of Tenebrio molitor in vitro

$\text{In vitro}$ organ cultures of corpora allata or corpora cardiaca-corpora allata complexes from $\text{Tenebrio molitor}$ were found to produce methyl-(2E, 6E)-(10R)-10, 11- epoxy-3, 7, 11 trimethyl-2, 6-dodecadienoate (JH III). No detectable JH I or II was produced. The hormone was identified by derivation, chromatography and mass spectral analysis. A ¹⁴C radiolabel was incorporated into the methyl carbon of the ester function from precursor L-[$\text{methyl}$-¹⁴C]-methionine added to the culture medium.     

The identification and biosynthesis of two juvenile hormones from the tobacco budworm moth (Heliothis virescens)

Brain-corpora cardiaca-corpora allata complexes from the tobacco budworm $\text{Heliothis virescens}$ produce both radiolabelled methyl (2E, 6E, 10Z)-10,11-epoxy-3, 11-dimethyl-7-ethyl-2, 6-tridecadienoate (JH I) and methyl (2E, 6E, 10Z)-10, 11-epoxy-3, 7, 11-trimethyl-2, 6-tridecadienoate (JH II) when cultured in medium containing L-[$\text{methyl}$−¹⁴C] methionine or sodium [1−¹⁴C] propionate. Degradative studies of the hormones derived from propionate show the specific incorporation of the latter into the homo-isoprenoid portions of these compounds.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocdrca gregaria.

We have compared, on an individual basis, the volume of the corpora allata with their ability to synthesize and release juvenile hormone (JH) using glands taken at daily intervals throughout the period of sexual maturation and the first two ovarian cycles in $\text{Schistocerca gregaria}$. A standard $\text{in vitro}$ radiochemical assay was used to measure the rates of both spontaneous JH biosynthesis from [methyl-¹⁴C]-methionine, and of JH biosynthesis stimulated by optimal concentrations of [C-2 ³H]-farnesenic acid. Computation of results showed that there are, during this period, changes of up to 250-fold in the rate of spontaneous JH biosynthesis per unit volume corpora allata. It is concluded that the volume of the corpora allata is of no value as an indicator of the spontaneous synthetic activity of the glands in this species, and that the overall rate of JH synthesis is regulated by mechanisms that do not involve large changes in the volume of the gland cells. However, in the presence of farnesenic acid, there is a corelation between stimulated JH synthesis and glandular volume, suggesting that the volume of the gland reflects the maximum activity of the final two stages in JH biosynthesis.     

Comparative metabolism of isoleucine by corpora allata of nonlepidopteran insects versus lepidopteran insects,in relation to juvenile hormone biosynthesis

We studied the metabolism of [U-¹⁴C]isoleucine by intact and homogenized corpora allata (CA) from various insect species to determine how this substrate is converted to precursors of juvenile hormone (JH). CA homogenates of the lepidopterans Manduca sexta, Hyalophora cecropia, and Samia cynthia metabolize [U-¹⁴C]isoleucine to several products including 2-keto-3-methyl-valerate, 2-methylbutyrate, CO₂, propionate, and acetate. Intact CA of male H. cecropia produce particularly high levels of 2-keto-3-methylvalerate, indicating a highly active branched-chain-amino acid transaminase. In contrast, CA homogenates from the nonlepidopterans Periplaneta americana, Schistocerca nitens, Tenebrio molitor, and Diploptera punctata barely metabolize [U-¹⁴C]isoleucine. However, P. americana CA homogenate metabolizes [U-¹⁴C]2-keto-3-methylvalerate, the transamination product of [U-¹⁴C]isoleucine, more rapidly than does a homogenate of M. sexta CA. Furthermore, intact CA from P. americana incubated with [U-¹⁴C]2-keto-3-methylvalerate incorporate low levels of ¹⁴C into JH III, but do not metabolize this substrate to JH II or JH I. Intact CA from female Diploptera punctata produce very high levels of JH III, but are also unable to incorporate radiolabel from [U-¹⁴C]isoleucine into JH III, which substantiates our findings with other nonlepidopteran CA. The results suggest that CA of nonlepidopteran insects lack an active branched-chain amino acid transaminase and, consequently, are unable to utilize these substrates for JH biosynthesis.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Enzymatic formation of 14,15-leukotriene A and C(14)-sulfur-linked peptides

Human leukocytes converted [³H]-(S)-15-HPETE into [³H]-14,15-LTA. Rat basophilic leukemia cells transformed 14,15-LTA into two bioactive C(14)-S-linked peptides, which have been characterized as 15(S)-hydroxy-14(R)-S-glutathionyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid and 15-(S)-hydroxy-14(R)-S-cysteinylglycyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid by comparison with synthetic specimens.     

The isolation and identification of juvenile hormone from cockroach corpora allata in vitro

Methyl (2E, 6E) - 10, 11 - epoxy - 3, 7, 11 - trimethyl - 2, 6 - dodecadienoate (JH III) is synthesized by corpora cardiaca/allata of the American cockroach $\text{Periplaneta americana in vitro}$ and released into the culture medium. It constitutes the principal part of the biologically detectable JH activity. A fully synthetic medium without terpenoid precursors or serum was used, which proves, that the entire molecule is synthesized $\text{de novo}$. The presence of JH III in orders as different as Blattodea, Orthoptera, Coleoptera and Lepidoptera indicates, that it is the most widely occuring JH of insects.     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Biosynthesis of (10R)-Juvenile hormone III from farnesoic acid by Aedes aegypti ovary

Synthesis of (10R)-juvenile hormone III (JH III) outside the corpora allata (CA) was investigated in female Aedes aegypti. Intact females or ligated abdomens of blood-fed and sugar-fed females synthesized in vivo [12-³H]JH III-like molecules from [12-³H]-methyl farnesoate, indicating that an organ(s) in the female abdomen, other than the CA, converted methyl farnesoate into JH III. To find out the organ(s) that synthesized JH III-like molecules, ovaries, fat bodies, and midguts were incubated in vitro with [12-³H]methyl farnesoate and the synthesis of JH III-like molecules was compared with JH III synthesized by CA. To identify tissue(s) having both farnesoic acid methyl transferase and farnesoate epoxidase, enzymes that convert farnesoic acid into JH III, ovaries, and fat bodies were removed from sugar and blood-fed females and incubated with [12-³H]farnesoic acid. Chemical derivatization by methoxyhydrin formation followed by esterification with (+)-α-methoxy- α-trifluoromethyl phenylacetic (MTPA) acid chloride and reversed phase liquid chromatography identified (10R)-JH III methoxyhydrin (+)-MTPA ester as the sole JH III-like molecule produced in tissue culture incubation of ovaries. Since only (10R)-JH III is produced and not racemic JH III, the oxidation of farnesoic acid must be enzymatically mediated. Ovaries and corpora allata of female A. aegypti also synthesized [³H,¹⁴C]JH III from L-[methyl-³H]methionine and [¹⁴C]acetate which was characterized by HPLC and gas chromatography. These results suggest that mosquito ovary can synthesize (10R)-JH III from farnesoic acid, and that this tissue synthesizes JH III-like molecules from L-methionine and acetate. © 1994 Wiley-Liss, Inc.     

Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro

When corpora allata from adult female $\text{Schistocerca}$$\text{gregaria}$ are incubated $\text{in}$$\text{vitro}$ with either ³H-$\text{trans}$, $\text{trans}$ farnesenic acid or ³H-$\text{trans}$, $\text{trans}$, $\text{cis}$ bishomo-farnesenic acid and [$\text{methyl}$-¹⁴C] methionine, they fabricate large quantities of the corresponding double labelled methyl 10, 11-epoxy esters. Radio GLC of these products indicates retention of geometric configuration at the C-2 and C-6 double bonds. Separate analyses of the contents of the glands and medium after incubation show that the epoxy esters are rapidly released from the glands into the medium and that only the glands contain the corresponding unepoxidized esters. We suggest that unepoxidized esters are the intracellular intermediates in the formation of juvenile hormones from the unsaturated acids. Gel filtration shows that the epoxy esters are not released as stable protein complexes but as simple solutes into the medium. Using this method of promoting the synthesis of juvenile-hormone-active compounds, rates of biosynthesis of epoxy esters of up to 33 ng. per pair of glands per hour have been achieved.     

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-¹⁴C]glucose ([¹⁴C]2dGlc) at various concentrations and the intracellular concentrations of [¹⁴C]2dGlc and deoxy[¹⁴C]glucose phosphate ($\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$) were measured. Using 7 μM extracellular [¹⁴C]2dGlc, the intracellular [¹⁴C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of $\text{2}\text{dGlc}\text{P}$. The rate of accumulation of total radioactivity ([¹⁴C]2dGlc plus $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$ decreased after 20 min using 7 μM extracellular [¹⁴C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [¹⁴C]2dGlc, but radioactivity disappearing from the cells was largerly $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [¹⁴C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM $\text{3-O-}\text{methylglucose}$, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of $\text{2}\text{dGlc}\text{P}$ and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.     

Nuclear binding sites for juvenile hormone III in the male accessory reproductive glands of Melanoplus sanguinipes

Summary

We have identified a potential nuclear juvenile hormone (JH) receptor in the long hyaline tubules (LHT), part of the male accessory reproductive gland (MARG) of M. sanguinipes. The MARG was incubated in vitro with [³H]JH III, and the distribution of the [³H]JH III among the cellular fractions of the LHT was determined. Some 37±4% of the radioactivity was associated with the crude nuclear pellet, while the cytosolic, microsomal and mitochondrial fractions contained 30±3%, 23±2% and 10±1%, respectively. The bound JH III was measured in nuclear extracts of LHT from males up to 15 days post-eclosion. These results revealed that JH binding increased in an age-dependent manner up to day 7, then levelled off to day 12, to increase again on day 14. The nuclear-binding component in the LHT had a very strong affinity for JH III, with a K_D value of 0.8 nM. Our observations are considered in relation to the potential site and mode of action of JH.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Juvenile hormone synthesis by ring glands of the blowfly Lucilia cuprina

The major radiolabelled product released from ring gland and brain-ring gland complexes of third instar larval and pre-pupal stages of the sheep blowfly Lucilia cuprina upon incubation with L-[methyl-³H]methionine corresponded to one diastereomer of juvenile hormone III bisepoxide (JHB₃). Endocrine glands incubated with the juvenile hormone precursor 2E,6E-farnesoic acid released increased quantities of JHB₃, together with significant amounts of juvenile hormone III but not the isomeric methyl 2E-6,7-epoxyfarnesoate. Synthesis of JHB₃ was developmentally and neurally regulated. Ring glands and brain-ring gland complexes from third instar larvae released more JHB₃ than comparable preparations from pre-pupae, while isolated corpus allatum segments of the gland were more active than intact brain-gland complexes. These results reinforce the emerging status of JHB₃ as the characteristic juvenile hormone of dipteran insects. Arch. Insect Biochem. Physiol. 34:239–253, 1997. © 1997 Wiley-Liss, Inc.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

Effects of the anti hormone-hormone mimic ETB on the induction of insect juvenile hormone esterase in Trichoplusia ni.

Juvenile hormone (JH) esterases can be artificially induced to appear in the hemolymph of last instar larvae of the lepidopterous insect $\text{Trichoplusia}$$\text{ni}$ (Noctuidae) by topical treatment with JH I, JH II, or dihomo branched juvenoids. ETB (ethyl-4-[2-(t-butylcarbonyloxy) butoxy] benzoate; ZR-2646) at high doses is a weak inducer of JH esterase (JHE). However, at doses of ETB that induce only low levels of JHE activity, ETB will block the JHE induction caused by the dihomo juvenoid epofenonane and at higher doses will reduce the induction caused by JH I or JH II. ETB is not a JHE inhibitor; rather, it appears to be acting as a JH agonist/antagonist in normal larvae and in isolated abdomens. These effects of ETB on JHE induction may illustrate a new mode of action of anti-JH's.     

Characteristics of leucine transport by isolated hepatocytes of antarctic fish at low temperatures

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [¹⁴C]leucine at 0, 5, and 10°C. The system is saturable with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0–10°C range yielded $\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 65}\text{kJ/mol}$ ($\text{Q}{}_{10}\text{= 2.7}$). The average first-order rate constant at 0°C was 0.1 min^?1, one-third the value of 0.3 min^?1 estimated for clearance of [¹⁴C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0°C and in temperate fish acclimated to 10°C and 20°C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.