Uptake of beta-ecdysone by the fat body and membrane vesicles of fat body cells of Sarcophaga peregrina larvae.

The fat body of Sarcophaga peregrina larvae was shown to incorporate ³H-β-ecdysone when it was incubated with the hormone in vitro. Most of the incorporated radioactivity was found in the cytoplasmic fraction as free β-ecdysone, not as a protein-β-ecdysone complex.Rapid uptake and accumulation of β-ecdysone was observed in the membrane vesicles of fat body cells in vitro. The apparent K_m value for uptake was estimated to be 1·25 × 10^?7 M. The β-ecdysone in the membrane vesicles was rapidly released when 2,4-dinitrophenol was added. These results suggest that β-ecdysone was incorporated into the membrane vesicles by active transport and not by free diffusion. The hormone is probably incorporated into larval tissues by the same mechanism as it is incorporated into the membrane vesicles of fat body cells.     

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.     

Induction of selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae by 20-hydroxyecdysone.

20-Hydroxyecdysone was shown to induce selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae with a molecular mass of 30 kDa. This phosphorylation was not associated with synthesis of new protein. Fractionation of 32P-labelled fat body by differential centrifugation showed that this protein was mainly present in the membrane-rich fraction, although we could not specify the membrane. Thus, 20-hydroxyecdysone may modify the function of the fat body by inducing phosphorylation of a specific membrane protein.     

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.     

Activation of the secretion of specific proteins from fat body following injury to the body wall of Sarcophaga peregrina larvae

When the body wall of Sarcophaga peregrina larvae was injured, the fat body was transiently activated to synthesize and secrete 70k and 27k proteins. It was found that genes for these proteins were activated at the time of the injury. The amount of resulting mRNA was too small to detect a translation product in vitro, using the reticulocyte lysate system, but the mRNA seemed to be translated quite efficiently in vivo, suggesting translational control of these mRNA.     

Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina.

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.     

Effect of the exotoxin of Bacillus thuringiensis on normal and ecdysone-stimulated ribonucleic acid polymerase activity in intact nuclei from the fat body of Sarcophaga bullata larvae

A nuclear preparation from the fat-body of Sarcophaga bullata was obtained which incorporates nucleotides at a steady rate. Two activities, differing in their response to alpha-amanitin, are present. The activities are not separated by changes in the concentration of (NH(4))(2)SO(4) as effectively as in mammalian nuclei. The activity resistant to alpha-amanitin is stimulated by ecdysone, and both normal and the ecdysone-stimulated activities are inhibited by the exotoxin. The amanitin-sensitive enzyme is also inhibited by exotoxin, but higher concentrations are required.     

Identification of 30-kDa fat body protein of Sarcophaga peregrina larvae selectively phosphorylated in the presence of 20-hydroxyecdysone as ribosomal protein S6

Previously, we showed that 20-hydroxyecdysone induces selective phosphorylation of a fat body protein of Sarcophaga peregrina with a molecular mass of about 30,000 (30-kDa protein) (Itoh, K., Ueno, K., & Natori, S. (1985) Biochem. J. 227, 683-688). This paper describes the identification of this 30-kDa protein. From the electrophoretic profile of 40S ribosomal proteins on two-dimensional polyacrylamide gel electrophoresis, the 30-kDa protein was identified as S6.     

Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury

A lectin was purified from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin agglutinated sheep red blood cells markedly and the hemagglutinating activity was inhibited by galactose and lactose. The active lectin was found to have a molecular weight of 190,000 and to consist of four alpha subunits and two beta subunits, with molecular weights of 32,000 and 30,000, respectively. During the early pupal stage, similar hemagglutinating activity in the hemolymph increased to several times than in larval hemolymph. This activity was completely inhibited by the antibody prepared against the lectin purified from the hemolymph of injured larvae. Thus, the same protein having lectin activity is apparently induced under two different physiological conditions: injury of the body wall of larvae and during pupation. The biological significance of this lectin is discussed.     

Purification of the 200 kDa hemocyte membrane protein of Sarcophaga peregrina and its specific interaction with fat body

A 200 kDa protein specifically expressed on the surface of pupal hemocytes of Sarcophaga peregrina was purified from the hemocyte membrane. This protein has been suggested to participate in dissociation of the fat body in the pupal stage of this insect. This protein was found to inhibit the dissociation of the fat body in vitro. Furthermore, it was shown to bind to the fat body and the binding could be saturated. These results suggested that pupal hemocytes expressing the 200 kDa protein interact directly with specific binding sites on the basement membrane of the fat body when they disintegrate this tissue.     

Participation of hemocyte proteinase in dissociation of the fat body on pupation of Sarcophaga peregrina (flesh fly)

In holometabolous insects, larval tissues are degraded on pupation. Previously, we established an in vitro system in which Sarcophaga fat body dissociates in the presence of pupal hemocytes, mimicking the situation in vivo (Kutata et al., J. Insect Physiol.35, 559–565, 1989). In this paper, we showed that chymostatin and phenylmethylsulfonyl fluoride prevented dissociation of fat body in this system. Moreover, of the various proteinases tested, only chymotrypsin degraded the fat body in vitro. These results suggested that a chymotrypsin-like proteinase of hemocytes participates in dissociation of the fat body at the early pupal stage.     

Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina.

When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.     

Activation of latent ribonuclease in the fat-body of fleshfly (Sarcophaga peregrina) larvae on pupation.

A crude extract of the fat-bodies of third-instar larvae of Sarcophaga peregrina (fleshfly) was found to contain latent RNAase (ribonuclease) consisting of RNAase and inhibitor protein that is sensitive to p-chloromercuribenzoic acid. The RNAase activity in the crude extract of fat-bodies became detectable with time after puparium formation, indicating that the inhibitor is selectively inactivated and RNAase is released from the RNAase-inhibitor complex during metamorphosis.     

DNA-dependent RNA polymerase I from human placental nuclei

This paper describes a large-scale method for solubilisation and purification of DNA-dependent RNA-Polymerase I from mature human placenta. The solubilisation method involves homogenization of the whole human placenta, isolation of cell nuclei, sonication of separated nuclei at high ionic strength and ammonium sulfate precipitation. The purification method consists of chromatography of RNA-Polymerase I activity on DEAE-Sephadex A-25 and Phosphocellulose P-11, and glycerol-density gradient centrifugation. In result, RNA-Polymerase I of human placenta nuclei has been shown to be completely resistant to alpha-amanitin. Besides dependence of RNA-Polymerase I on different Mg2+ and Mn2+ concentrations, glycerol concentration and ionic strength was studied. Using our results, an optimal RNA-Polymerase I assay mixture was developed. The subunit composition of RNA-Polymerase I was investigated by dodecylsulfate-gel electrophoresis. The RNA-Polymerase I molecule of human placenta consists of 13-14 polypeptides.     

Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.     

Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae.

Three antibacterial proteins were induced when the body wall of Sarcophaga peregrina (flesh-fly) larvae was injured with a hypodermic needle. These proteins were separated and one was purified to homogeneity. The molecular weight of the purified protein was 5000 and its amino acid composition was similar to that of cecropins, which are antibacterial proteins in Hyalophora cecropia (cecropia moth) pupae. This protein was found to have bactericidal activity and to be effective at a concentration of 0.1 micrograms/ml against certain Gram-negative and Gram-positive bacteria.     

Induction of pupal diapause and photoperiodic sensitivity during early development of Sarcophaga peregrina larvae

Effects of long day and short day treatments during the embryonic and larval stages on induction of pupal diapause were studied on a diapausing race of Sarcophaga peregrina. Two long day (15L 9D) cycles during 2 days before or after the larviposition completely stopped the induction of pupal diapause on larvae which grow in short day condition before and after the long day treatment. The sensitivity appeared to decrease during the early stage of the third instar and to increase again to some extent in the prepupal stage.     

Intracellular freezing, viability, and composition of fat body cells from freeze-intolerant larvae of Sarcophaga crassipalpis.

Although it is often assumed that survival of freezing requires that ice formation must be restricted to extracellular compartments, fat body cells from freeze-tolerant larvae of the gall fly, Eurosta solidaginis (Diptera, Tephritidae) survive intracellular freezing. Furthermore, these cells are highly susceptible to inoculative freezing by external ice, undergo extensive lipid coalescence upon thawing, and survive freezing better when glycerol is added to the suspension medium. To determine whether these traits are required for intracellular freeze tolerance or whether they are incidental and possessed by fat body cells in general, we investigated the capacity of fat body cells from nondiapause-destined and diapause-destined (i.e., cold-hardy) larvae of the freeze-intolerant flesh fly Sarcophaga crassipalpis (Diptera, Sarcophagidae) to survive intracellular freezing. Fat body cells from both types of larvae were highly susceptible to inoculative freezing; all cells froze between -3.7 to -6.2 degrees C. The highest rates for survival of intracellular freezing occurred at -5 degrees C. The addition of glycerol to the media markedly increased survival rates. Upon thawing, the fat body cells showed little or no lipid coalescence. Fat body cells from E. solidaginis had a water content of only 35% compared to cells from S. crassipalpis larvae that had 52-55%; cells with less water may be less likely to be damaged by mechanical forces during intracellular freezing.