Cecropia Research Articles

Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori

1. 1. Cecropins, inducible antibacterial peptides, were purified by simple two step chromatography from immunized larval hemolymph of the silkworm, Bombyx mori. 2. 2. The cecropins were further separated into four major and two minor forms by reverse-phase HPLC. 3. 3. The four major cecropins were sequenced and divided into two types, A and B, whose structures were quite similar to cecropins A and B of Hyalophora cecropia. 4. 4. Three of them contained an unusual amino acid, δ-hydroxylysine. 5. 5. No remarkable difference in antibacterial activity against E. coli was detected among these cecropins.

Assessment of antibiotic potentials of insect antibacterial factors

Immune haemolymphs from the giant silkmoth, Hyalophora cecropia and, to a lesser extent, from the tsetse, Glossina morsitans morsitans possess antibacterial activities against several species of bacteria known to be pathogenic to man, animals and poultry, as demonstrated by in vitro bacterial growth inhibition assays. Cecropia immune haemolymph possesses a broader antibacterial spectrum and was found to be active against several Gram-positive and Gramnegative pathogenic bacteria. Mice injected with pre-determined lethal doses of Enterobacter cloacae, Klebsiella pneumoniae and Corynebacterium pseudotuberculosis and then treated with single intraperitoneal or subcutaneous injections of Cecropia immune haemolymph had much lower mortality than untreated controls. A possibility of developing a broad-spectrum antibiotic modelled on insect immune factors is discussed.

Exine structure in Cecropia L. (Moraceae) pollen grains

Devido ao pequeno tamanho dos grãos de pólen de espécies do gênero Cecropia, é elucidada no presente trabalho a estratificação da exina e a ornamentação das superfícies por meio de observações em microscopia eletrônica. As 10 espécies estudadas podem ser separadas em dois grupos segundo a distribuição dos espículos na superfície dos grãos de pólen: distribuição uniforme com espículos acumulados na faixa equatorial, deixando as áreas polares praticamente lisas. A exina é estruturada de fora para dentro por espículos supratectais, de um teto bem desenvolvido atravessado por diminutos canalículos, báculos curtos e delgados, uma nexina 1 descontínua e uma nexina 2 contínua, espessada em volta dos poros. De acordo com estas diferenciações da estrutura da exina, o gênero Cecropia apresenta dentro da família Moraceae um certo grau de evolução que não apoia a sua aproximação à família Urticaceae.

Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga. Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.

Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation

Prophenoloxidase (PPO) has been purified to homogeniety from hemolymph of Hyalophora cecropia. There are two forms of the enzyme with identical molecular weights (76 kDa). Four proteins directly involved in the activation of PPO have also been purified from the hemolymph. Active phenoloxidase is elicited by the addition of factor C1 and a serine protease (SPII), which alone cannot activate PPO. Purified SPII contains two proteins with M r 43 and 53 kDa, the larger molecule may represent the unactivated enzyme. An inhibitor of the SPII catalyzed activation of PPO has been isolated. In addition a protein presumed to be dopa quinone imine conversion factor has been purified.

Solid state 13C nuclear magnetic resonance and chemical analyses of insect noncuticular sclerotized support structures: Mantid oothecae and cocoon silks

Sclerotized oothecae from the praying mantids, Stagmomantis carolina and Tenodera sinensis, and cocoon silks from the moths, Antheraea mylitta, A. polyphemus, Hyalophora cecropia, Bombyx mori, Plodia interpunctella and Ephestia cautella, were subjected to solid state 13C nuclear magnetic resonance and chemical analysis. The oothecae are composed of protein (83% of the wet wt), water (7–8%), diphenolic compounds (6%) and inorganic salts (2–3%). The major diphenols extracted in cold dilute acid are N- acylated derivatives of dopamine and norepinephrine, including N- malonyldopamine , N- acetyldopamine and N- acetylnorepinephrine . The acid-extractable diphenols account for <1% of the total diphenols estimated by solid state NMR analysis, indicating that nearly all of the diphenols in mantid egg cases are involved in covalent cross-links or adducts with oothecal proteins. The silks are lower than the oothecae in diphenolic compounds (0.1–1%), slightly higher in protein (88–90%) and about the same in water (7–8%) and inorganic salt content (3–4%). A higher proportion of the total diphenols is acid extractable from the silk (4–56%) than from the oothecae, and they include 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxyphenylalanine, 3,4-dihydroxybenzaldehyde and N- acetylnorepinephrine . Subsequent metabolism of diphenols in these structures probably involves oxidation to quinonoid derivatives, followed by formation of covalent bonds between diphenol ring or side chain carbons and nucleophilic groups in the structural proteins.

Investigation of an Ortho-quinone isomerase from larval cuticle of the American silkmoth, Hyalophora cecropia

Insect cuticles catalyze the formation of N-acetylnorepinephrine (NANE) and N- β-alanylnorepinephrine (NBANE) from N-acetyldopamine (NADA) and N- β-alanyldopamine (NBAD), respectively. An enzyme, involved in the reaction, has now been isolated from fifth stage larval cuticle of Hyalophora cecropia and partially characterized. The enzyme alone has hardly any activity towards NADA, but together with diphenoloxidases [catechol oxidases (EC 1.10.3.1) or laccases (EC 1.10.3.2)] it will produce NANE as the main product from NADA, indicating that NADA-quinone is the actual substrate for the enzyme. The enzyme is presumably an ortho-quinone para-quinone methide isomerase, and formation of NANE is due to non-enzymatic addition of water to the quinone methide. The enzyme combination mushroom tyrosinase-cuticular isomerase has pH optimum at 5.5, and the optimal substrate concentration is about 10 mM NADA. Together with the endogenous cuticular diphenoloxidases the isomerase can account for the formation of NANE observed when pieces of intact cuticle are incubated with NADA, and for the presence of NANE and NBANE in sclerotized cuticle. The possible roles of the enzyme in sclerotization and defense reactions in insects are briefly discussed.

Kinetics of yolk precursor uptake in Hyalophora cecropia: Stimulation of microvitellin endocytosis by vitellogenin

Ovarian follicles of Hyalophora cecropia selectively incorporate several extracellular proteins into yolk. In order to determine how the proportions of these proteins in the yolk are established two of them, vitellogenin and microvitellin, were presented to vitellogenic follicles in vitro. When the two were presented to follicles together, they did not compete for the uptake mechanism, suggesting separate binding sites. On the contrary, vitellogenin accelerated the uptake of microvitellin by a factor of two. It had a similar effect on fluid phase uptake, and therefore appears, unlike microvitellin or any other protein tested, either to enhance the rate of endocytotic activity or to suppress resecretion during membrane recycling. Estimates of K uptake lead to the conclusion that vitellogenin saturates its endocytotic mechanism in situ, but microvitellin may not.

The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study.

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.

Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes

A genomic clone was isolated from the tobacco hornworm, Manduca sexta, by virtue of its similarity to a Drosophila larval cuticle gene. RNA analysis shows that this clone, B311, is expressed at times appropriate for a larval cuticle gene. Hybrid-selection experiments using B311 DNA show that it encodes a 14 × 10 3 M r protein, LCP-14, which is precipitated by an antiserum to Manduca larval cuticle. We have sequenced both genomic and cDNA clones for the LCP-14 gene. A conceptual translation of the cDNA sequence shows that the LCP-14 protein is similar not only to another Manduca cuticle protein, but also to Drosophila, Sarcophaga and Hyalophora cecropia cuticle proteins. Since these proteins are found in flexible cuticle and have similar sequences, we conclude they are encoded by homologous genes.

Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes.

Cecropins, positively charged antibacterial peptides found in the cecropia moth, and synthetic peptide analogs form large time-variant and voltage-dependent ion channels in planar lipid membranes in the physiological range of concentration. Single-channel conductances of up to 2.5 nS (in 0.1 M NaCl) were observed, which suggests a channel diameter of 4 nm. Channels formed by the peptides cecropin AD and MP3 had a permeability ratio of Cl-/Na+ = 2:1 in 0.1 M NaCl. A comparative study of the three cecropins, cecropins A, B, and D, and of six synthetic analogs allowed determination of structural requirements for pore formation. Shorter amphipathic peptides did not form channels, although they adsorbed to the bilayer. A flexible segment between the N-terminal amphipathic region and the C-terminal more hydrophobic region of the peptide was required for the observation of a time-variant, voltage-dependent conductance. Cecropin AD was the most effective voltage-dependent pore-forming peptide and was also the most potent antibacterial peptide against several test organisms. A positive surface charge or cholesterol in the bilayer reduced the conductances caused by cecropin AD or MP3 by at least 5-fold. This behavior is consistent with the known insensitivity of eukaryotic cells to cecropins. Our observations suggest that the broad antibacterial activity of cecropins is due to formation of large pores in bacterial cell membranes.

Anomalous behaviour of 86Rb as a tracer for transintestinal potassium transport in the fowl, Gallus domesticus.

ABSTRACT Fluxes of potassium ions across biological membranes can be measured using 42K as a tracer. This isotope, however, has a half-life of only 12-4 h and it is expensive. Rubidium belongs to the alkali metal series of elements and rubidium ions behave in many respects, physicochemically and biologically, like K+. Furthermore, 86Rb has a half-life of 18-7 days and is less expensive than 42K. Thus 86Rb has been used quite widely as a tracer for estimating K+ transport. In mammals this usage has included erythrocytes (Beauge &amp; Adragna, 1971), the crystalline lens (Becker, 1962), renal tubules (Ellison, Velazquez &amp; Wright, 1986) and large intestine (Tannen, Marino &amp; Dawson, 1986; Freel, 1987). In these instances, the ratios of the measured fluxes, JK/JRb were not found to differ significantly from unity. In non-mammals, 86Rb has been shown to be a useful substitute for K+ in transport studies using flounder intestine (Frizzell et al. 1984), salmon erythrocytes (Bourne &amp; Cossins, 1984) and frog skin (Zerahn, 1983). In fish gills, the ratio JK/JRB was consistently found to be about 1·3 (Sanders &amp; Kirschner, 1983), thus allowing for correction of estimates of K+ flux based on the use of 86Rb as a tracer. In the American silkworm (Hyalophora cecropia) gut, this ratio was 1·1 in a flat-sheet preparation (Wood &amp; Harvery, 1979), but in a spherical preparation it was rather unpredictable with a mean of 1 -7 (Zerahn, 1980). None of these authors considered 86Rb to be a reliable substitute for 42K. Eddy (1985) has drawn attention to such possible disparities of K+ metabolism estimated by the use of 86Rb in fish. We have also found differences in estimates of K+ transport using 42K and 86Rb as tracers in the avian intestine.

The structure of the gene for cecropin B, an antibacterial immune protein from Hyalophora cecropia

Pupae of the moth Hyalophora cecropia respond to an injection of live bacteria by the production of a potent antibacterial activity. The broad-spectrum property of this activity is due chiefly to two small proteins, cecropins A and B. Sequences of the proteins showed them to be homologous and to contain 37 and 35 amino acid residues respectively. The subsequent isolation of two cDNA clones for cecropin B showed that this protein is made as a prepro molecule composed of 62 amino acid residues. We have now prepared a genomic bank and studied four genomic clones for cecropin B. The coding regions were found in two neighbouring BglII fragments, one 0.79 kb and another varying in size from 3.1 kb to 4.9 kb for different clones. One transcriptional unit for preprocecropin B was sequenced and found to be 1035 bp long with a single intron, 514 bp in size. A conserved, insect specific cap site, ATCATTC, was identified by S1 mapping and primer extension experiments. Indications were found for the presence of multigene families and multicopy genes.

Potassium channel blockers unmask electrical excitability of insect follicles

AbstractStandard techniques for intracellular recording and current injection were used to determine if the developing ovarian follicles of six species of insect were electrically excitable. Follicles were dissected from the panoistic ovarioles of Locusta migratoria and Periplaneta americana, the polytrophic meroistic ovarioles of the fruit fly Drosophila rnelanogaster and the silkworm moths Hyalophora cecropia and H. Columbia, and the telotrophic meroistic ovarioles of the mealworm beetle Tenebrio mlitor. Follicles of all species except Tenebrio were inexcitable in control saline; Tenebrio follicles produced short action potentials which did not outlast the duration of the current pulse. Follicles of all species except Drosophila produced long duration overshooting action potentials when depolarized in salines containing the potassium channel blockers tetraethylammonium (TEA; 10–80 mM), Bazf (10–20 mM) and 4‐aminopyridine (4‐AP; 10 mM). Hyalophora follicles were also excitable in saline containing TEA and Ba2+ but not 4‐AP, and Tenebrio follicles were excitable in saline containing Ba2+ but not TEA and 4‐AP. Excitability in follicles of all species was unaffected by sodium replacement but was reversibly inhibited by the calcium channel blockers La3+ (1 mM), Co2+(10 mM), or verapamil (0.1‐1.0 mM). It is suggested that the action potentials are caused by the movement of calcium or barium through voltage‐dependent channels that are masked in control saline by a large counteracting potassium conductance. Excitability appears to be a property of the oolemma and not the follicle cell membranes. Possible functions of voltage‐dependent ion channels in insect oocytes are discussed.

Adsorptive endocytosis of vitellogenin, lipophorin, and microvitellogenin during yolk formation in Hyalophora

AbstractYolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody‐binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.

Synthesis of immune proteins in primary cultures of fat body from Hyalophora cecropia

Fat body from injured or immunized pupae as well as from untreated pupae of Hyalophora cecropia synthesize and release in vitro the immune proteins P4, attacins, lysozyme and cecropins. During an incubation time of 4 days immune protein synthesis declines relative to the other proteins, i.e. protein synthesis returns to a normal pupal pattern. Treatment of fat body in culture with bacterial compounds (LPS, lyophilized cells of Micrococcus lysodeikticus or heat killed Escherichia coli), but also sterile filtered insect Ringer, causes a relative higher rate of synthesis of the immune proteins. At the same time synthesis of some large proteins (75–100 kD) is switched off. Similar results are obtained by adding hemocytes from injured or immunized pupae, whereas hemocytes from untreated pupae show no effect. The induction of protein synthesis with particular emphasis on the immune proteins will be discussed.

Effects of ionophores on vitellogenin uptake by Hyalophora oocytes

AbstractOocytes of Hyalophora cecropia that were incubated in vitro with [35S]vitellogenin incorporated label within 10 min into an intermediate‐density compartment identified by sucrose density gradient centrifugation. During a subsequent 20‐min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m‐cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K+ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H+‐K+ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.

Insect immunity: cDNA clones coding for the precursor forms of cecropins A and D, antibacterial proteins from Hyalophora cecropia

Using synthetic probes cDNA clones were isolated correponding to the precursor forms of cecropins A and D. The sequences obtained were compared to earlier data for preprocecropin B. A processing scheme in three or four steps is discussed.

Cell-free immunity in insects.

Abstract An inducible antibacterial activity is a main part of the immune system of the Cecropia moth and several other insects. The active material is a family of small proteins called cecropins. Sequence work shows that at least two of the cecropins originate from a gene duplication. Work on the biosynthesis has been initiated on RNA and tissue levels.

Ecdysteroid action on moth epithelial tissues and cell lines

AbstractThe spontaneous formation of multicellular, fluid‐filled vesicles in primary cultures of pupal wings of Hyalophora cecropia is inhibited by 20‐hydroxyecdysone (20‐OH‐ecdysone). Physiological levels of ecdysteroids inhibit the ability of larval hemolymph to induce such vesicles in an established cell line (IAL‐TND1) derived from imaginal discs of Trichoplusia ni (Hübner). This cell line grew as vesicles for its first year in culture even in the absence of hemolymph, but then converted to growing as multicellular aggregates. Aggregate‐conditioned media but not vesicle‐conditioned or control media displayed ecdysteroid activity in an imaginal disc bioassay. Larval hemolymph had no influence on the imaginal discs' response to ecdysteroids. Hence, its action in promoting vesicle formation is not to prevent the action of ecdysteroids. We postulate that the change in morphology of TND1 cell cultures from vesicles to aggregates was accompanied by their attaining (or enhancing) the capacity to produce ecdysteroids.