保幼激素的代谢

保幼激素的代谢由保幼激素酯酶、保幼激素环氧水解酶和保幼激素二醇激酶等共同催化完成。在这些代谢酶的作用下,保幼激素代谢成保幼激素酸、保幼激素二醇、保幼激素酸二醇和保幼激素二醇磷酸。作者总结了保幼激素代谢的研究方法;按实验室和昆虫种类为线索,归纳和概括了每一种保幼激素代谢酶的研究进程;对保幼激素酯酶和保幼激素环氧水解酶作了序列分析;最后对保幼激素的代谢研究进行了展望。     

Nuclear development in locust fat body: the influence of juvenile hormone on inclusion bodies and the nuclear matrix

The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components greater than 150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

Regulation of glycolytic intermediates in cockroach fat body by the corpus cardiacum

Glycolytic intermediates and related metabolites were measured in the fat body of the American cockroach (Periplaneta americana) to locate the rate-limiting reactions that regulate glycolysis during the action of the corpus cardiacum (CC) in vitro.

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.     

Phospholipid synthesis in the fat body endoplasmic reticulum during primary and secondary juvenile hormone stimulation of vitellogenesis inLeucophaea maderae

Summary Juvenile hormone (JH) treatment coordinately stimulated the dose-dependent synthesis of vitellogenin and endoplasmic reticulum (ER) membrane phospholipids in fat body cells from allatectomized adult females ofLeucophaea maderae. Animals were pulse-labeled in vivo with [³²P] to simultaneously measure the rates of synthesis of the phosphorylated subunits of vitellogenin and the structural phospholipids of the ER membranes. Phospholipid synthesis in ER membranes from nontarget tissues for JH such as thoracic muscle, midgut, and larval fat body was unresponsive to hormone treatment. The proliferation of ER in response to JH treatment was thus restricted to tissue that was competent to synthesize vitellogenin.Primary and secondary vitellogenin induction was measured in allatectomized adult females treated 12 days apart with JH-III. The time-course of the primary response for vitellogenin and ER phospholipid synthesis was characterized by a 24 h latent period, a rapid increase to a maximum at 72 h, and then a gradual decline. During secondary induction, vitellogenin accumulated in the hemolymph nearly twice as fast as before and peaked at a concentration of 38 g/l. This vitellogenin titer was approximately two-fold higher than that found at the height of the primary response. During both primary and secondary stimulation with JH, ER phospholipid synthesis, as measured by [¹⁴C]choline incorporation into microsomal phosphatidylcholine, was stimulated five-fold over the untreated control animals. The amplified production of vitellogenin during the secondary response was associated with a 24 h-earlier peak of ER phospholipid synthesis in the fat body.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Regulation of phospholipase A(2) activity in cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone: evidence for the participation of protein kinase C

Phospholipase A₂ (PLA₂) associated with the membrane fraction of trophocytes from Periplaneta americana fat body increases by as much as 100% when the cells are incubated with hypertrehalosemic hormone (HTH-II). Activation with HTH-II is approximately halved by inclusion of the PKC inhibitor sphingosine in the incubation medium. Because activation of PLA₂ by HTH-II is blocked by the GDP analogue GDP-β-S, and the unactivated enzyme is activated by the GTP analogue GTP-γ-S it is likely that a G protein is involved in activation of the enzyme. Activation of PLA₂ was also achieved by treating the trophocytes with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol in the presence of thapsigargin. This supports the view that protein kinase C is also involved in the activation process.     

Comparison of some properties of the high-affinity juvenile hormone binding protein from the larval hemolymph of pyralid moths

The properties of the high-affinity low molecular weight juvenile hormone (JH) binding protein present in the hemolymph of larvae of five species of pyralid moths, a noctuid moth, and a sphingid moth were compared. The pyralid moths exhibit a facultative diapause as last-instar larvae. The species employed were the southwestern corn borer, Diatraea grandiosella, the southern cornstalk borer, Diatraea crambidoides, the sugarcane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, the sunflower moth, Homoeosoma electellum, the cabbage looper, Trichoplusia ni, and the tobacco hornworm, Manduca sexta. The binding characteristics of the proteins were determined using saturation binding assays and competitive binding assays. The dissociation constants of JH I, JH II, and JH III for the binding protein of all the species varied from 0.8 x 10^?7 M to 2.8 x 10^?7 M. Calibrated gel filtration showed that the binding protein of all the species had apparent molecular weights ranging from 29,000 to 31,000. Electrophoresis in 7% acrylamide gels revealed that the relative mobilities of the binding proteins ranged from 0.33 to 0.43. Isoelectric focusing showed that the binding proteins had isoelectric points between 4.4 and 5.0.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

Stimulation of vitellogenin production by methoprene in prepupae and pupae of manduca sexta

Denaturing electrophoresis of hemolymph from prepupae of M. sexta showed trace amounts of polypeptides with mobilities corresponding to those of vitellogenin (Vg) apoproteins from adult females. Absence of the polypeptides in allatectomized insects suggested regulation by juvenile hormone (JH). Daily administration of 10 μg of the JH analog methoprene from day 4 of the fifth stage to day 0 of the pupal stage caused accumulation of these polypeptides. They were identified as apovitellogenins (apoVgs) immunochemically with Vg antiserum. Stimulation of Vg in response to methoprene varied with age. In all cases, day 0 female pupae were highly responsive. Vg synthesis was not stimulated when pupae were injected with 20-hydroxyecdysone (20-HE) in addition to methoprene. Methoprene-stimulated Vg synthesis was also abolished by inhibitors of mRNA or protein synthesis (α-amanitin, actinomycin, cycloheximide). This result indicated that methoprene-stimulated Vg accumulation requires gene expression. A Vg cDNA (2.1 kb) obtained by immunoscreening of the λgt 11 library, when used as a radiolabelled probe, hybridized with a 5.1 kb mRNA from total RNA of female fat body. It also hybridized with fat body RNA of normal prepupae and methoprene treated day 0 pupae but not with that of early fifth instars or solvent control pupae. The results indicate that the trace amounts of Vg found in prepupal stages are due to a weak expression of the Vg gene, which is stimulated by JH and repressed by 20-HE. © 1994 Wiley-Liss, Inc.     

Presence and regulation of trehalose in the body of adult Phormia regina

Trehalose, the major blood sugar of Phormia regina, is present within its tissues in an amount exceeding that in the total blood volume. A major part of the reserve is found in the abdominal fat body. An investigation of trehalose regulation, pursued with the use of a trehalose tolerance test, indicates that within a period of 4 h the adult fly can remove from its blood amounts of this sugar in excess of twice its normal level. The surplus is dealt with in an as yet unknown way, being either sequestered in the tissues (not as trehalose or glucose), metabolized, or excreted in a form other than trehalose or glucose. The process is regulated by the head, and a link between the body and the head must be maintained throughout the entire period of activity.     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body: Cytological development

Cytological development in the fat body of adult female Locusta migratoria, related to vitellogenin synthesis, has been studied by light and electron microscopy. In the newly-emerged adult, the cells are filled with lipid droplets, which indent the nucleus, and with fields of glycogen, while ribosomes and endoplasmic reticulum are scarce. Correlated with the onset of vitellogenin synthesis, about day 8 of adult life, the nucleus enlarges, lipid droplets and glycogen decrease, and rough endoplasmic reticulum and Golgi complexes become the most abundant organelles. These changes reflect a conversion of the principal role of the fat body from nutrient storage to the synthesis and secretion of protein. They are prevented by allatectomy, and restored by subsequent treatment with the juvenile hormone analogue, ZR-515. Late in the first gonotrophic cycle, about day 20, dense bodies, vesicle-containing bodies and lysosomes are seen, indicating recycling of cellular materials. Five days after ZR-515 treatment, when protein synthesis has declined, the rough endoplasmic reticulum appears in arrays adjacent to lipid droplets, possibly awaiting reactivation. By the use of ferritin-labelled antivitellin immunoglobulin, vitellogenin has been localized intracellularly in the RER saccules and Golgi vesicles, and extracellularly in channels between the folded plasma membranes, showing sites of accumulation and secretion of this protein.     

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate‐binding motifs were predicted in the protein. Modeling of the 3‐D structure showed that the protein consisted of eight α‐helixes linked with loops, with no β‐sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

The role of juvenile hormone esterase in terminating larval feeding and initiating metamorphic development in Trichoplusia ni

This study shows, first, that when JH degradation by JHE is blocked with an inhibitor (EPPAT, O-ethyl-S-phenyl-phosphoramidothiolate), prothoracicotropic/ecdysone release/effects are postponed in the cabbage looper Trichoplusia ni (Hübner) (Noctuidae). Thus, JHE is an important component of JH degradation, implying that without normal degradation the JH titer will become abnormally high. Second, this accumulation of endogenous JH in EPPAT treated larvae results in an extra larval molt. Therefore, JHE is also important in the control of the nature of the molt, by controlling the JH titer. Third, this study demonstrates that EPPAT at proper doses is a viable probe for studying enzyme and hormone action in vivo without pharmacological artifacts.

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Résumé Cette étude indique d'abord que, lorsque la dégradation de l'hormone juvénile (JH) par la JHE est bloquée par un inhibiteur (EPPAT, O-éthyl-S-phényl-phosphoramidothiolate) les effets prothoracicotropiques—libération d'ecdysone—sont retardés chez Trichoplusia ni Hübner. Ainsi, la JHE est un élément important de la dégradation de JH, impliquant que sans une dégradation régulière, la teneur en JH deviendra anormalement élevée. Cette accumulation d'hormone juvénile endogène chez les larves traitées à l'EPPAT provoque de plus une mue larvaire supplémentaire. Par conséquent, JHE est importante aussi dans le contrôle de la nature de la mue, en déterminant la teneur en JH. Enfin, cette étude a montré que l'EPPAT à des doses appropriées est un moyen efficace pur étudier l'action hormonale in vivo sans artéfacts pharmaceutiques.

     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

保幼激素类似物对斜纹夜蛾核型多角体病毒在宿主血淋巴中增殖动态的影响

通过研究保幼激素类似物(juvenile hormone analogues, JHA) methoprene对斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）在宿主血淋巴中增殖的影响,以探明JHA促进 SpltNPV增殖的初步机制,为阐明JHA促进病毒增殖提供更全面的理论依据。应用SDS PAGE及Western blot法,分析了methoprene对SpltNPV多角体蛋白(polyhedron, POLH)在宿主斜纹夜蛾6龄幼虫血淋巴中合成的影响。结果表明：经methoprene处理后2～3天可明显促进幼虫血淋巴液中POLH的合成。在此基础上,通过荧光定量PCR检测,发现methoprene对SpltNPV在幼虫血淋巴液复制的影响主要发生在处理后的第4和第5天,该期间polh基因的拷贝数比对照显著增加,拷贝数的峰值达1.22×10¹⁰/mL。