Factors influencing cyclic AMP and diacylglycerol levels in fat body of Locusta migratoria

Forskolin (FORSK), octopamine (OA) and adipokinetic hormone (AKH) stimulate the production of diacylglycerol (DG) in fat body of the locust, Locusta migratoria and the release of DG from fat body into hemolmph. The three effectors also increase the level of cAMP in fat body, but the cAMP content is not proportional to DG production. AKH stimulates the uptake of Ca²⁺ by fat body cells and requires the presence of extracellular Ca²⁺ to increase cAMP and DG levels in fat body. The production of DG seems to be an energy-dependent process. The uptake of DG by lipophorin (LP) from fat body is also energy-dependent but does not require extracellular Ca²⁺.     

Crystallization and preliminary analysis of crystals of apolipophorin III isolated from Locusta migratoria

Crystals of apolipophorin III, isolated from the locust Locusta migratoria, have been reproducibly grown from ammonium sulfate solutions and are well suited for an x-ray crystallographic analysis. Locust apolipophorin III is a glycosylated protein with a molecular weight of 19,100 and interacts with lipophorin, the major lipoprotein complex in insects. The crystals belong to the space group P6122 or P6522 with unit cell dimensions of a = b = 67.5 A, c = 155.6 A and diffract to a nominal resolution of 2.5 A. They are physically robust and are stable in the x-ray beam for over a week. A complete native x-ray data set has been collected and processed to 3.0-A resolution.     

Antifeedants against Locusta migratoria from the Japanese Cedar, Cryptomeria japonica II

A methanol extract of Cryptomeria japonica completely inhibited feeding by Locusta migratoria. Based on bioassay-guided fractionation, two active terpenols, (+)-ferruginol and (-)-cubebol, were isolated and identified as antifeedants against this insect species. Each compound separately showed weak activity, but they showed intense activity against this insect species when they were combined.     

Hydrolysis of glycerides by lipases of the fat body of the locust,Locusta migratoria

Lipolysis of mono- di- and triacylglycerol by locust fat body preparation was studied. Microsomes and the soluble supernatant contained an acid lipase (pH optimum 5.0–6.0) which hydrolized micellar dispersions of the above lipids. On short incubations, diacylglycerol yielded equimolar amounts of monoacylglycerol and fatty acids. Trioleylglycerol was degraded very slowly; mainly fatty acids were formed. The enzyme was inhibited by detergents, and various salts.Monoacylglycerol was hydrolyzed by the supernatant over a pH of 5.5 to 7.5. In the microsomes the activity continued to increase up to pH 9.0. These activities were not inhibited by Triton X-100. Moreover, at pH 7.8 in the presence of Triton, di- and triacylglycerol were also degraded.     

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo.

Biosynthetic processes related to the production of vitellogenin (yolk precursor protein) have been examined in the fat body of adult female Locusta migratoria. Vitellogenin-producing capacity was assayed by incubation of fat body with [³H]leucine, followed by precipitation from the medium with specific antiserum. In normal development, vitellogenin synthesis began at about Day 7 after emergence and became maximal at about Day 13, when this protein accounted for 60% of the total fat body protein output. The production of other proteins increased to a lesser extent, becoming maximal at about Day 6. The incorporation of uridine into fat body RNA rose to a maximum at Day 8, which coincided with a marked increase in tissue RNA content. The DNA content in adult female fat body approximately doubled between Days 3 and 8. Vitellogenin synthesis, and the increases in RNA and DNA, were prevented by removal of the corpora allata (the source of juvenile hormone). In allatectomized locusts, vitellogenin synthesis was induced by JH or an analog, ZR-515. Applied topically in acetone, these gave steep dose-response curves, half-maximal at 75 and 150 μg, respectively. After a single treatment with ZR-515, fat body vitellogenin production rose slowly during 48 hr, then steeply to a maximum at 72 hr, but after decay of this effect during 10 days, a second application of ZR-515 induced renewed synthesis with little initial lag. Hormone treatment produced a smaller increase in the output of other proteins, and an increase in incorporation into RNA which preceded the major rise in vitellogenin synthesis. Male fat body produced little or no vitellogenin. These results are consistent with action of JH at the gene level.     

Different isoforms of an apoprotein (apolipophorin III) associate with lipoproteins in Locusta migratoria

Insects transport lipid for flight in the form of diacylglycerol-rich low-density lipoproteins (low-density lipophorin, LDLp), which in the hemolymph are produced from high-density lipophorin (HDLp) by reversible association with several molecules of an apolipoprotein, apolipophorin III (apoLp-III, Mr approximately 18,000-20,000) during lipid loading. Two isoforms of apoLp-III (a and b) were purified both from adult Locusta migratoria migratorioides hemolymph and LDLp, which have identical apparent Mr but differ in amino acid composition, NH2-terminal amino acid sequence, and isoelectric points (5.35 +/- 0.01 for apoLp-IIIa, 5.10 +/- 0.01 for apoLp-IIIb). The NH2-terminal sequence of apoLp-IIIb is identical to the primary structure of apoLp-III deduced from cloned cDNA [Kanost et al. (1988) J. Biol. Chem. 263, 10,568-10,573], whereas the NH2-terminal sequence of apoLp-IIIa is identical to that of apoLp-IIIb but preceded by Arg-Pro-, which is the C-terminal of the putative signal peptide coded by cDNA upstream from that coding for apoLp-IIIb. The ratio apoLp-IIIa apoLp-IIIb free in hemolymph is identical to that in LDLp (5:9); since 14 molecules of apoLp-III appear to be bound in one molecule of LDLp, an average of 5 molecules of apoLp-IIIa and 9 of apoLp-IIIb are involved in formation of each LDLp particle. In vivo studies using 35S-labeled apoLp-IIIa and b demonstrate that each of the isoforms can associate with HDLp to produce LDLp reversibly; in an in vitro system, production of LDLp containing exclusively apoLp-IIIa or apoLp-IIIb demonstrates independent participation of each isoform in LDLp formation.     

Antifeedants against Locusta migratoria from the Japanese Cedar, Cryptomeria japonica

(1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one and (+)-7(14),10-bisaboladien-1-ol-4-one were isolated and identified from Cryptomeria japonica as antifeedants against Locusta migratoria L. which is well known as a serious pest to cereals throughout the world. These compounds strongly inhibited the feeding of L. migratoria only when they were combined, but each compound alone did not show any activity.     

Antifeedants against Locusta migratoria from the Japanese Cedar,Cryptomeria japonica

(1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one and (+)-7(14),10-bisaboladien-1-ol-4-one were isolated and identified from Cryptomeria japonica as antifeedants against Locusta migratoria L. which is well known as a serious pest to cereals throughout the world. These compounds strongly inhibited the feeding of L. migratoria only when they were combined, but each compound alone did not show any activity.     

Differential regulation of protein biosynthesis at translational level in the fat body cells of adult Locusta migratoria

Vitellogenin (Vg) and lipophorin (Lp) are synthesized by the fat body of adult locust (Locusta migratoria) females. We have shown by an immunohistochemical technique that both proteins are produced in the same cells of the fat body. The rate of Vg synthesis was measured with the use of double immunoprecipitation of labeled proteins at oviposition and 24 h later. It was found that the rate of Vg synthesis declined significantly by the time of oviposition; however, 24 h later, it was raised to the highest possible level. The rate of Lp synthesis remained constant at both indicated points. The similar postlaying increase in the Vg synthesis rate was observed in the fat bodies of females treated by alpha-amanitin immediately after oviposition. The data provide evidence that Vg biosynthesis in L. migratoria is regulated by selective periodical repression and derepression of Vg mRNAs in the fat body cells but not by total inhibition and stimulation of protein-synthesizing machinery.     

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ? adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Octopamine uptake systems in thoracic ganglia and leg muscles of Locusta migratoria

1. The octopamine uptake systems of the metathoracic ganglion and the extensor tibiae muscle of Locusta migratoria consists of a saturable Na⁺-dependent and an insaturable Na⁺-independent component.2. The Na⁺-dependent uptake component of the metathoracic ganglion consists of more than one subunits (K_D of the high affinity subunit 1.7 μmol), whereas the respective component of the extensor tibiae muscle does not (K_D = 60 μmol).3. Pharmacological investigations have demonstrated distinct differences in the profiles of the uptake between both tissues.4. Autoradiographic studies show that the main octopamine uptaking structures of the metathoracic ganglion are concentrated in the peripheral soma region. The extensor tibiae muscle, however, has no specialized uptake region.     

X-linkage of a vitellogenin gene in Locusta migratoria

Hybridization of a cloned DNA probe to blots of restriction digests of Locusta migratoria genomic DNA showed that a vitellogenin gene is present at one copy per haploid genome in females, and at only one-half that number in males. The genomic region encompassing the 3′-coding end of the gene is polymorphic, as revealed by blots of DNA from individual locusts. DNA from some female locusts yielded two variants in this region, whereas a series of male locusts showed one variant or the other, but never both. The results demonstrate that the vitellogenin gene, which is normally expressed only in females, is X-linked in L. migratoria.     

Membrane-cytoskeleton interactions: Inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.