ISSN:0739-4462    

DOI:10.1002/arch.940300202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAntisera were raised against different domains of a putative ecdysteroid receptor (cEcRH) of Chironomus tentans. All the antisera reacted with a 68,000 dalton protein exhibiting DNA binding properties. Additionally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [3H]ponasterone A, with high specificity. These properties indicate that the antisera recognize specifically an endogenous ecdysteroid receptor protein (cEcR) in C. tentans cells and thus are suitable for the following quantitative and qualitative immunological and immunohistochemical investigations. The cellular level of cEcR varies during development, and it is particularly low in oligopausing larvae. In polytene chromosomes of prepupal salivary glands, cEcR is located at approximately 50 transcriptionally active loci. These loci include both early ecdysteroid (Ec)‐inducible puff sites, such as the locus containing the gene coding for the homolog of the E75 protein in Drosophila melanogaster, as well as late Ec‐inducible puff‐sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultraspiracle protein. However, loci of genes coding for salivary gland secretory proteins (e.g., Balbiani ring forming chromosome regions) do not specifically react with the antisera. Thus, the developmental regulation of these genes is not directly controlled by Ec. Polytene chromosomes of oligopausing larvae show hardly any loci that contain cEcR. The few detected correspond, with few exceptions, to the most potent cEcR binding sites found in prepupae. © 1995 Wiley‐L

ISSN:0739-4462    

DOI:10.1002/arch.940300203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe activities of RNA polymerases I and II in the fat body of the tobacco hornworm larva increased during feeding and then declined during wandering in the fifth instar (Sridhara and Gilbert, Dev Biol 45:7–21 [1975]). RNA polymerase II activity was much higher than that of RNA polymerase I. To determine how the differences in the activities of the two enzymes and their developmental changes correlate with changes in RNA and protein, the contents of these macromolecules were determined during the same period. RNA and protein increased by about 8 to 10‐fold during the feeding period. Activities of RNA polymerases I and II in nuclei, as well as those present in “free” (not transcribing) and “engaged” (transcribing) pools, were measured. Incorporation of labeled uridine into RNA by fat body cells in vitro was correlated to nuclear enzyme activities. The amounts of RNA polymerases I, II, and III were determined by the application of an ELISA with subunit‐specific monoclonal antibodies. Results showed that even though the in vitro activity of RNA polymerase II and its amounts are higher than those of RNA polymerase I and III, rRNA and tRNA syntheses predominated over mRNA synthesis in vivo. The activities and amounts of RNA polymerase II in the “free” pool were higher than those for RNA polymerase I. The “free” pools of the two enzymes increased as the larva progressed through the fifth instar. There appears to be little correlation between RNA polymerase activities and RNA synthesis during wandering, as RNA synthesis by the tissue and nuclear RNA polymerase activities declined faster than the amounts of the enzymes. The amount of RNA remained fairly steady during the same period. 20‐hydroxyecdysone did not affect either the RNA polymerase activities or RNA synthesis of the fat body cultured in vitro.

ISSN:0739-4462    

DOI:10.1002/arch.940300204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAdult functions of juvenile hormone (JH) have been described forDrosophila melanogasterand other dipteran insects, but preadult function for this hormone remains largely unknown in this order of insects. We have identified a mutation ofDrosophila, Methoprene‐tolerant (Met), which appears to alter JH reception during late larval development. The molecular cloning ofMetwill be a step toward understanding this gene and possibly identifying a preadult role(s) for JH. Molecular cloning was initiated using the technique of transposon‐tagging with a transposablePelement.P‐element insertional alleles ofMetwere generated, and genomic libraries were constructed from two of these alleles. From these librariesP‐element‐bearing clones were isolated that in situ hybridized to the cytogenetic region whereMethad been previously localized by genetic methods. Two of the alleles were shown to have completeP‐elements inserted in similar, but not identical, locations in the predicted cytogenetic region whereMetis located. A late‐larval cDNA library was screened to identify transcriptional units in this region, and clones were recovered with homology to a DNA fragment abutting theP‐element insertion site. These clones may representMetcDNA molecules. © 1995

ISSN:0739-4462    

DOI:10.1002/arch.940300205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAn assay was developed with which to study basic characteristics of an adenylyl cyclase in the corpora allata (CA) of the tobacco hornworm,Manduca sexta.The assay used glands collected and frozen at −80°C, to circumvent the problem of tissue availability. With this protocol for storage of tissue, less than 25% of the enzyme activity in fresh tissue was lost. Substances such as sodium fluoride (NaF) and Gpp(NH)p (a non‐hydrolyzable GTP analog), which typically stimulate the adenylyl cyclases in other insect tissues, increased enzyme activity several‐fold. There was a progressive decrease in the capacity of the CA adenylyl cyclase to be stimulated by NaF during the fifth stadium, suggesting a possible developmental change in the capacity of the associated G protein to be stimulated by NaF. The calcium/calmodulin (CaM) dependence of adenylyl cyclase activity was also investigated. The results demonstrated that addition of up to 10−4M calcium to assays of enzyme activity in whole gland homogenates of both larval (day O) and prepupal (day 6) CA resulted in only a slight increase in the activity of the enzyme over basal rates in the presence of the calcium chelator EGTA. However, addition of as little as 5 m̈M CaM in the presence of 10−4to 10−3M calcium increased adenylyl cyclase activity three to five‐fold. A similar stimulation was obtained with washed membrane preparations of day 0 and day 6 glands, but required a substantially higher concentration of CaM. Results demonstrated that the CA possess a calcium/CaM‐dependent adenylyl cyclase from day 0 through day 6. A preliminary investigation of the effect of two biogenic amines on the CA adenylyl cyclase revealed that enzyme activity was not affected by octopamine, but a stage‐specific effect was obtained with dopamine. Concentrations of 10−6and 10−7M stimulated enzyme activity in hornogenates of day 0 glands but inhibited activity in homogenates of day 6 CA.

ISSN:0739-4462    

DOI:10.1002/arch.940300206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractNumerous studies have demonstrated regulation of specific lepidopteran proteins by pharmacological doses of insect juvenile hormone (JH). In this study, topical application of a 1 pg dose of JH I to fourth stadium larvae of theblack(bl) mutant strain of the tobacco hornworm,Manduca sexta, induced a 50% increase in the titer of hemolymph juvenile hormone binding protein (hJHBP). Radioimmunoassay confirmed that JH titers were lower inbllarvae than in wild‐type larvae at the time of JH treatment. Enzyme immunoassay analysis of hJHBP titers demonstrated that regulation by JH I was dose‐dependent at doses up to 10 pg and that the response was saturated above 100 pg. Western blotting and equilibrium dialysis confirmed these results and demonstrated that hJHBP frombllarvae had the same molecular mass and displayed the same affinity for JH I as hJHBP isolated from wild‐type larvae. Time course studies showed that regulation was complex: 1 2 h after JH I treatment, hJHBP titers were twofold lower in treated than in controlbllarvae, while 44 h after treatment they were twofold higher. JH I regulation of hJHBP titers inbllarvae was independent of changes in total hemolymph protein. © 1995 Wiley‐L

ISSN:0739-4462    

DOI:10.1002/arch.940300207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBaculovirus insecticides are receiving renewed attention as insect pest control agents following the development of fast‐acting recombinant baculoviruses. Here we report on the construction and biological activity of a recombinant baculovirus derived from the nuclear polyhedrosis virus ofAutographa californicawhich expresses a modified form of juvenile hormone esterase (JHE). The serine at the catalytic site of the JHE has been mutated to a glycine residue so that the protein does not degrade JH. The recombinant baculovirus expressing this modified form of JHE, named AcJHE‐SG, has enhanced activity against lepidopteran larvae. Lethal times of the recombinant are 20 to 30% lower than for the wild type virus, and a 66% reduction in feeding damage caused by infected larvae is observed. This result is comparable to the best recombinant baculovirus developed to date, AcAaIT, which expresses an insect‐selective scorpion toxin. The potential of these recombinant viruses for commercialization as insecticides is discussed. Bioassays of AcJHE‐SG in conjunction with anti‐JH agents indicate that the virus is not killing by an anti‐JH mechanism. Larvae apparently die from contraction‐paralysis, or disruption of the normal sequence of events at the molt. © 1995 W

ISSN:0739-4462    

DOI:10.1002/arch.940300208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractTheapterous (ap)gene inDrosophilamelanogaster encodes a homeodomain transcription factor. It is required for the development of the wings and of a subset of embryonic muscles. The gene has been implicated in the juvenile hormone (JH) system because mutations inaplead to JH deficiency, and are associated with defective histolysis of the larval fat body, arrested vitellogenesis, sterility, and aberrant sexual behavior, all of which are dependent on JH.We describe here the use of hemizygotes and germ‐line clones, of X‐ray‐and hybrid dysgenesis‐induced lethalapalleles to determine the primary role of the gene during development. We find thataplethality is polyphasic, but occurs primarily at the larval and pupal stages. The lethal phenotype is not associated with any overt morphological abnormality, suggesting that death occurs from a systemic malfunction. Strong interallelic complementation for the wing phenotype was found between someapmutations induced by hybrid‐dysgenesis. By Northern blot analysis, we demonstrate an increase inapexpression in pupae and adults as compared to embryos and larvae, suggesting that it is developmentally regulated. Finally, primer extension is used to determine the transcription start site of the gene. © 1995 Wiley

ISSN:0739-4462    

DOI:10.1002/arch.940300209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractChemical modification procedures were employed to neutralize charged amino acid side chains of apolipophorin III (apoLp‐III). Glutamate plus aspartate carboxylate side chains were amidated while, in other experiments, the ϵ‐amino groups of lysine residue side chains were acetylated. Circular dichroism (CD) spectroscopy was performed to assess the effect of chemical modification on the secondary structure of apoLp‐III. Compared to control, unmodified apoLp‐III, both amidated and acetylated apoLp‐IIIs possessed significantly diminished levels of α‐helical structure. A similarly significant amount of α‐helix structure could be induced in both modified apoLp‐IIIs, however, by the addition of 50% trifluoroethanol, a helix inducing solvent, indicating that the proteins have retained their capacity to form helical secondary structures. The lipid binding interactions of chemically modified apoLp‐IIIs were also examined in lipoprotein binding assays. Whereas control, unmodified apoLp‐III displayed lipid binding activity, neither modified apoLp‐III was capable of interaction with the substrate lipid surface. In phospholipid binding assays using the model compound, dimyristoylphosphatidylcholine, acetylated apoLp‐III failed to interact while amidated apoLp‐III showed limited interaction. When sodium dodecyl sulfate (SDS) micelles were employed as a model lipid surface, interaction of the modified apoLp‐IIIs was observed. To characterize the relative stability of the interaction of control and modified apoLp‐IIIs with SDS micelles, urea denaturation studies were performed. These experiments showed that, while control and amidated apoLp‐IIIs were relatively resistant to urea induced denaturation, acetylated apoLp‐III was susceptible. Taken as a whole, the results suggest that charged amino acid residues play an important role in stabilization of the lipid‐free helix bundle conformation of apoLp‐III and may promote stabilization of the lipid bound state through charge‐charge interactions with lipoprotein

ISSN:0739-4462    

DOI:10.1002/arch.940300210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAdult femaleDrosophila melanogastercorpus allatum (CA) synthesize JHB3from endogenous and exogenous precursors in vitro. We present evidence supporting the thesis that biosynthesis proceeds from precursor FA via initial epoxidation and terminal methylation on the basis of the following: (1) Methyl farnesoate is not epoxidized to JHIII or JHB3; (2) Authentic JHIII is not epoxidized to JHB3; and (3) FABE is markedly metabolized to JHB3. Cerebral allatostatic factors act at some stage subsequent to FA and this precursor is not normally rate‐limiting. Additionally, neural inhibition from the brain acts at some biosynthetic step prior to FA. © 1995 Wiley‐Liss,

ISSN:0739-4462    

DOI:10.1002/arch.940300211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY