Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

Dependence of juvenile hormone activity on haemolymph factor in Tenebrio molitor pupae

Morphogenetic response of Tenebrio molitor pupae to juvenile hormone (JH) varies with age, it being low during the first 6 hr after pupal ecdysis and reaching a peak at about 18 hr after ecdysis. Parabiosis of 6-hr pupa to a 18-hr-old pupa causes a higher morphogenetic response in 6-hr pupa to JH. A similar increase in morphogenetic response was not noticeable when a 0·45 μm Millipore filter was placed between the parabiotic partners, suggesting that the factor(s) may be macromolecular. This view is further supported by the observation that the Millipore filter did not prevent transport of ecdysterone another agent that enhances morphogenetic response of pupae to JH.     

The effect of juvenile hormone on protein synthesis in the transparent accessory gland of male Rhodnius prolixus

Feeding stimulates the accumulation of protein by the transparent accessory reproductive glands (TARG), allatectomy abolishes the increase, and severing the nervous connections between the corpus allatum (CA) and the brain raises the level of accumulation after feeding. TARG removed from allatectomised females exhibit a markedly decreased level of incorporation of [³H]-leucine into protein in vitro, and topical application of juvenile hormone (JH) to allatectomised males restores the ability of the glands to incorporate [³H]-leucine in vitro. Severing the nerves between the CA and the brain increases the rate of incorporation in vitro, but in both operated and normal males, the rate of incorporation decreases with time after feeding. JH applied topically to intact animals during the falling phase of incorporation, or applied directly to TARG in vitro from such animals, stimulates incorporation. These data indicate that JH from the CA controls protein synthesis in the TARG and that the CA in the male of Rhodnius may be subject to different controls than in the female.     

Control of cuticle formation by wing imaginal discs in vitro.

Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [³H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro.     

Disturbance of Adult Eclosion by Fenoxycarb,a Juvenile Hormone Mimic,in the Beet Armyworm,Spodoptera exigua

Effect of exogenous juvenile hormones (JHs) on pupal development was assayed in the beet armyworm, Spodoptera exigua. Fenoxycarb, a potent JH mimic, was applied topically to different ages of the pupae, and showed significant inhibition of normal adult eclosion even at 0.1 μg dose when it was applied at the early pupal stage (day 0). As the pupal development underwent, the susceptibility of the pupae fenoxycarb decreased. RH5992, a potent ecdysteroid mimic, did not, however, any similar inhibitory effect on the pupae. Natural JH types (JH I, JH II, and JH III) were applied on day 0 pupae to compare their inhibitory effects on adult eclosion. Both JH I and JH II significantly inhibited adult eclosion at 1.0 μg dose, but JH III did not even at 10.0 μg dose. It was noted that fenoxycarb-treated pupae showed little rectum development. Fenoxycarb did not, however, show any negative effect on the development of compound eye and wing imaginal discs, and on the pupal hemolymph protein pattern. These results suggest that there should be a commitment period requiring an absence of JH for a normal adult metamorphosis during early pupal development and that the endogenous type of JH in S. exigua is JH I or JH II or both JHs like other lepidopteran species.     

Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age

Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed.The synergistic effect of 1 μg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to >18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter's ability to synchronize epidermal cells.The significance of these observations in the analysis of time of action of juvenile hormone is discussed.     

Release of a juvenile hormone binding protein by fat body of the Indian meal moth, Plodia interpunctella,in vitro

When fat body of fifth instar larvae of Plodia interpunctella was cultured in vitro in a chemically defined medium, the tissue released a low mol. wt protein (FBBP) that binds juvenile hormone (JH). This FBBP has the same mol. wt, estimated by gel permeation chromatography, as the haemolymph JH binding protein. Furthermore, the FBBP protected JH from degradation by general esterases isolated from the haemolymph. Treatment of fat body with cycloheximide inhibited incorporation of [¹⁴C] leucine into the FBBP protein fraction and reduced the amount of FBBP released into the medium. We conclude that one source of the JH binding protein found in the haemolymph is the fat body.     

Biogenesis of mammalian mitochondria in the renoprival kidney. I. Amino acid incorporation and respiratory activity

Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [³H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation.     

Bacteria, antibiotics and amino Acid incorporation into maize endosperm protein bodies

The incorporation of C¹⁴-leucine into protein bodies isolated from developing maize endosperm was studied. Protein bodies which incorporated significant amounts of C¹⁴-leucine were found to be contaminated with bacteria, while protein bodies prepared with sterile techniques incorporated C¹⁴-leucine at very low rates.

Streptomycin, penicillin, puromycin, and actinomycin did not completely inhibit the bacterial incorporation, while chloramphenicol was an effective inhibitor.

     

The control of chitin deposition by ecdysterone in larvae of Bombyx mori

The effect of ecdysterone on the deposition of chitin in Bombyx larvae was examined. The chitin content in the abdomen decreased following hormone treatment to a value half that of the controls. Studies with ¹⁴C-glucose revealed that whereas controls exhibited a gradual decrease in the rate of ¹⁴C-glucose incorporation into chitin, the administration of ecdysterone resulted in a rapid fall in the rate of incorporation followed by a rise until ecdysis occurred. Chitin catabolism was estimated at 0·11 mg chitin/hr, based on the chitin content and incorporation rates. A dual rôle is indicated for ecdysterone in chitin metabolism, namely the activation of both synthetic and lytic systems.     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly,Luciola lateralis

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.     

Interaction between leucine and palmitate catabolism in 3T3-L1 adipocytes and primary adipocytes from control and obese rats

Metabolic profiling studies have highlighted increases in the plasma free fatty acid (FFA) and branched-chain amino acid (BCAA) concentrations, which are hallmarks of the obese and insulin-resistant phenotype. However, little is known about how the increase of the BCAA concentration modifies the metabolic fate of FFA, and vice versa, in adipocytes. Therefore, we incubated differentiated 3T3-L1 adipocytes or primary adipocytes from rats fed a control or a high-fat diet with: (1) 0, 250, 500 and 1000 μM of leucine and determined the oxidation and incorporation of [1-¹⁴C]-palmitate into lipids or proteins or (2) 0, 250, 500 or 1000 μM of palmitate and evaluated the oxidation and incorporation of [U-¹⁴C]-leucine into lipids or proteins. Leucine decreased palmitate oxidation and increased its incorporation into the lipid fraction in adipocytes; the latter was reduced in adipocytes from obese rats. However, palmitate increased leucine oxidation in adipocytes as well as reduced leucine incorporation into the protein and lipid fractions in adipocytes from obese rats. These results demonstrate that leucine modifies the metabolic fate of palmitate, and vice versa, in adipocytes and that the metabolic interaction between leucine and palmitate catabolism is altered in adipocytes from obese rats.     

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.     

Effects of farnesyl methyl ether on the haemolymph and ovarian proteins of the tent caterpillar, Malacosoma americanum

The mode of antigonadotropic action of farnesyl methyl ether (FME) on Malacosoma americanum was investigated by studying the haemolymph and ovarial proteins during vitellogenesis. Low doses of FME which permitted apparently normal adult development but inhibited ovarian development were used.As indicated by the incorporation of ¹⁴C-glycine, FME treatment had no effect on the protein biosynthetic activity by the fat body. However, it resulted in significant accumulation of several haemolymph proteins apparently caused by their reduced uptake by the ovarioles.Radioassay of individual proteins revealed that all the haemolymph proteins had incorporated ¹⁴C-glycine. However, proteins G and H, and to some extent protein B of the treated females, showed significantly higher incorporation of the labelled glycine. It is concluded that the antigonadotropic action of the JH mimic was due to reduced incorporation of various sex-specific and non-specific haemolymph proteins into the ovarioles.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.

     

Protein degradation in silkworm peritracheal athrocytes and its physiological role in metamorphosis

The major functions of silkworm peritracheal athrocytes (nephrocytes) include endocytosis. Although athrocytes are also believed to function in protein degradation, there is limited experimental evidence for this. In this study, we detected the uptake and degradation of foreign proteins in peritracheal athrocytes by immunohistochemical, Western blot, and ex vivo analyses. IgG-FITC was detected in the athrocytes of silkworm larvae following injection, and LysoTracker analysis showed endosomal and lysosomal colocalizations. Athrocytes from larvae injected with IgG were incubated in Grace's medium for 2 days before being analyzed for the degradation of IgG by Western blotting. The level of incorporated IgG decreased and degradation products appeared following ex vivo culture. The highest level of IgG incorporation and degradation in the athrocytes was observed at the early pupal stage. The athrocytes also incorporated arylphorin, a major larval haemolymph protein and storage protein in silkworms. At the early pupal stage, arylphorin was actively degraded in the athrocytes. These results indicate that, in cooperation with the fat body, peritracheal athrocytes may function in the digestion of arylphorin during silkworm metamorphosis.