Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by beta-ecdysone.

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Molecular cloning and characterization of SRAM, a novel insect rel/ankyrin-family protein present in nuclei

Previously, we purified a 59-kDa protein that binds to the kappaB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the kappaB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SRAM and Dif were co-transfected, although SRAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei.     

Humoral factor activating the Sarcophaga lectin gene in cultured fat body

On culture of the fat body of Sarcophaga peregrina (flesh fly) larvae in modified Grace's medium, the Sarcophaga lectin gene was activated only when the medium was supplemented with larval hemolymph. The expressions of the genes for the storage protein and the sarcocystatin A were not affected by supplementing the medium with the hemolymph. Thus the hemolymph contained a factor that specifically activated the Sarcophaga lectin gene. This factor seemed to be a heat-stable, low molecular weight compound that was probably not a peptide, and to activate genes in the fat body for defense proteins that are known to be expressed in response to injury of Sarcophaga larvae.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Activation of the Sarcophaga lectin gene promoter in transgenic Drosophila

We established transgenic Drosophila strains in which the lacZ gene was expressed under the control of the 5'-upstream regulatory region of the Sarcophaga lectin gene promoter (3.1 kbp). The reporter gene was expressed in the fat bodies of the transgenic larvae when they were immunized by body pricking or treatment with Escherichia coli, which was the same as the Sarcophaga lectin gene expression in Sarcophaga larvae. However, the same reporter gene was found to be expressed constitutively in the digestive tracts of the transgenic larvae even without immunization.     

Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall

A previous paper described the purification of a lectin induced in the hemolymph of larvae of Sarcophaga peregrina (flesh-fly) on injury of their body wall (Komano, H., Mizuno, D., and Natori, S. (1980) J. Biol. Chem. 255, 2919-2924). This paper describes cDNA cloning and the complete nucleotide sequence of the gene for Sarcophaga lectin. Although active lectin consists of alpha and beta subunits in a molar ratio of 2:1, the fat body of injured larvae was found to contain only mRNA for the alpha subunit, suggesting that these two subunits are derived from a common gene and that the alpha subunit is converted to the beta subunit post-translationally. The alpha subunit was found to consist of 260 amino acid residues with an additional signal sequence of 19 or 23 amino acid residues.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Cationic glutathione S-transferase of human erythrocytes has unique kinetic characteristics among human glutathione S-transferases

The cationic glutathione S-transferase (GST sigma) of human erythrocytes is activated when incubated with 1 mM N-ethylmaleimide or other sulfhydryl blocking agents. Other GST isoenzymes of human tissues were inhibited by these reagents under similar conditions. At higher concentrations of NEM, GST sigma was also inhibited. Dithiothreitol, 2-mercaptoethanol, and sodium borohydride also caused several fold activation of GST sigma but noe of the other human GST isoenzymes were activated by these reagents.     

Identification and activation of storage protein receptor of Sarcophaga peregrina fat body by 20-hydroxyecdysone

Previous work showed that 20-hydroxyecdysone activates the fat body of Sarcophaga peregrina larvae to incorporate storage protein selectively from the hemolymph. In this study, storage protein receptors of the fat body membrane which were induced on pupation or on treatment of larval fat body with 20-hydroxyecdysone in vitro were identified. The binding of storage protein to its receptor required divalent cations, especially Ca2+, and the binding was very sensitive to pH. The storage protein receptor was inactivated when the fat body membrane was treated with trypsin. The storage protein receptor is probably a protein and it may be synthesized de novo or a cryptic form may be converted to the active form when the concentration of 20-hydroxyecdysone in the hemolymph reaches a physiological level.     

Molecular cloning of cDNA for sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina

A cDNA clone for sapecin, an antibacterial protein produced by an embryonic cell line of Sarcophaga peregrina, was isolated and characterized. This clone was found to encode a precursor of sapecin consisting of 94 residues, with sapecin (40 residues) constituting its carboxyl-terminal half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the hemocytes of the third instar larvae of Sarcophaga in response to body injury. Thus, sapecin is probably a defense protein synthesized by Sarcophaga to prevent bacterial infection through the damaged body wall. This gene was also found to be activated in the embryonic and early pupal stages, suggesting that sapecin also plays a role in the ontogenetic processes of Sarcophaga.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.