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A M Fallon - One of the best experts on this subject based on the ideXlab platform.

  • characterization of genomic dna encoding Cecropins from an aedes albopictus mosquito cell line
    Insect Molecular Biology, 2002
    Co-Authors: A M Fallon
    Abstract:

    We used cDNA probes from Aedes albopictus mosquito Cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions –192 to –165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells.

  • Cloning and expression of three cecropin cDNAs from a mosquito cell line.
    FEBS letters, 1999
    Co-Authors: E D Eccleston, A M Fallon
    Abstract:

    We have characterized full-length cDNAs encoding three isoforms of the antibiotic cecropin secreted by the C7-10 cell line from the mosquito, Aedes albopictus. The existence of two cecropin isoforms that differed from the previously described AalCecA was predicted by mass spectrometry and amino acid sequence analysis of peptides that eluted from reversed phase high performance liquid chromatography as a single peak just behind the previously described cecropin, AalCecA. Based on the amino acid sequence of the mature AalCecA peptide, we designed primers that amplified partial cDNAs encoding three different A. albopictus Cecropins in reverse transcriptase polymerase chain reactions. Rapid amplification of cDNA ends was then used to complete the cDNA sequences of AalCecA, AalCecB and AalCecC, respectively. Each cDNA encoded a translation product containing a signal peptide, a pro region, and a mature cecropin peptide consistent with amino acid sequence data from chymotryptic digests. Although the mosquito Cecropins shared 70-86% identity among each other, they shared only approximately 40% identity to Cecropins from Drosophila melanogaster. Each of the Cecropins was expressed within 2 to 4 h after induction, and transcripts measuring 0.3 to 0.5 kb continued to accumulate over 24 h. The three Cecropins were secreted in roughly equimolar proportions, and 30 to 90% of AalCecB was amidated at the terminal glycine residue. In contrast, amidated forms of AalCecA and AalCecC constituted a smaller proportion of these isoforms.

  • Cloning and expression of three cecropin cDNAs from a mosquito cell line
    FEBS Letters, 1999
    Co-Authors: E D Eccleston, A M Fallon
    Abstract:

    We have characterized full-length cDNAs encoding three isoforms of the antibiotic cecropin secreted by the C7–10 cell line from the mosquito, Aedes albopictus. The existence of two cecropin isoforms that differed from the previously described AalCecA was predicted by mass spectrometry and amino acid sequence analysis of peptides that eluted from reversed phase high performance liquid chromatography as a single peak just behind the previously described cecropin, AalCecA. Based on the amino acid sequence of the mature AalCecA peptide, we designed primers that amplified partial cDNAs encoding three different A. albopictus Cecropins in reverse transcriptase polymerase chain reactions. Rapid amplification of cDNA ends was then used to complete the cDNA sequences of AalCecA, AalCecB and AalCecC, respectively. Each cDNA encoded a translation product containing a signal peptide, a pro region, and a mature cecropin peptide consistent with amino acid sequence data from chymotryptic digests. Although the mosquito Cecropins shared 70–86% identity among each other, they shared only ∼40% identity to Cecropins from Drosophila melanogaster. Each of the Cecropins was expressed within 2 to 4 h after induction, and transcripts measuring 0.3 to 0.5 kb continued to accumulate over 24 h. The three Cecropins were secreted in roughly equimolar proportions, and 30 to 90% of AalCecB was amidated at the terminal glycine residue. In contrast, amidated forms of AalCecA and AalCecC constituted a smaller proportion of these isoforms.

  • Peptide Sequence of an Antibiotic Cecropin from the Vector Mosquito,Aedes albopictus
    Biochemical and Biophysical Research Communications, 1998
    Co-Authors: E D Eccleston, A M Fallon
    Abstract:

    Abstract We have identified a 35-amino acid antibiotic cecropin secreted by an established mosquito cell line. C7-10 cells from the vector mosquito,Aedes albopictus,were incubated with heat-killedEscherichia coli,and materials secreted into the cell culture supernatant were recovered by acid precipitation. Following batch elution from Sep-Pak C18 cartridges and further purification by reverse phase high performance liquid chromatography (RP-HPLC) a predominant peak of antibacterial activity was characterized by mass spectrometry, amino acid composition analysis, and Edman degradation, yielding the sequence GGLKKLGKKLEGVGKRVFKASEKALPVAVGIKALG. Unlike other Cecropins, the peptide was not amidated at the C-terminus.Aedes albopictusCecropin A (AalCecA) is the first cecropin to be described from a mosquito vector of human disease. Consistent with the classification of mosquitoes among the Dipteran suborder Nematocera, AalCecA shares only 36% amino acid identity with Cecropins fromDrosophila melanogasterand other Cyclorrhaphid flies, whose mature Cecropins share 80% to 100% amino acid identity.

Isao Morishima - One of the best experts on this subject based on the ideXlab platform.

  • Parallel induction of cecropin and lysozyme in larvae of the silkworm, Bombyx mori
    Developmental and Comparative Immunology, 1995
    Co-Authors: Isao Morishima, Tomoki Horiba, Masao Iketani, Eri Nishioka, Yoshiaki Yamano
    Abstract:

    Lysozyme activity in the hemolymph of Bombyx mori increased in parallel with cecropin activity after injection of the larvae with soluble peptidoglycan or UV-killed bacteria. The lysozyme and cecropin A genes were expressed in parallel in the fat body after injection of peptidoglycan as detected by northern blot hybridization. The elicitor specificity for lysozyme induction was identical to that for cecropin, suggesting a common mechanism for recognition of bacteria and following signal transduction introducing to the simultaneous synthesis of cecropin and lysozyme. Bacterial cells killed by UV-irradition were also effective as elicitor when added to the fat body culture, suggesting that phagocytosis of bacteria by hemocytes may not be an essential process for the induction of antibacterial protein synthesis in the silkworm.

  • cloning of cdnas for Cecropins a and b and expression of the genes in the silkworm bombyx mori
    Bioscience Biotechnology and Biochemistry, 1994
    Co-Authors: Yoshiaki Yamano, Masahito Matsumoto, Kenji Inoue, Tomohisa Kawabata, Isao Morishima
    Abstract:

    cDNA clones coding Cecropins A and B were isolated from a cDNA library constructed from the fat body of immunized Bombyx mori larvae. The cloned cDNAs had an open reading frame of 63 amino acids, indicating the primary translated peptides were processed to form mature Cecropins of 35 amino acid residues. The homology in the coding regions of Cecropins A and B was 73%.In immunized fat body, the expression of both cecropin A and B genes reached the maximal level 5 h after the injection of soluble peptidoglycan, and the high level was maintained until 9 h after immunization. The cecropin A and B genes were expressed at high levels in fat body and hemocytes, at lower but significant levels in malpighian tube, slightly in midgut, and none in silk gland.

  • isolation and structure of Cecropins inducible antibacterial peptides from the silkworm bombyx mori
    Comparative Biochemistry and Physiology B, 1990
    Co-Authors: Isao Morishima, Shigeyuki Suginaka, Teruo Ueno, Hisashi Hirano
    Abstract:

    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.

Yusuke Kato - One of the best experts on this subject based on the ideXlab platform.

  • cecropin p1 and novel nematode Cecropins a bacteria inducible antimicrobial peptide family in the nematode ascaris suum
    Biochemical Journal, 2005
    Co-Authors: Ajitha Pillai, Satoshi Ueno, Hong Zhang, Yusuke Kato
    Abstract:

    Cecropin P1 was first identified as a mammalian antimicrobial peptide isolated from the pig intestine. Much research aimed at characterizing this peptide has been reported. Recently, the workers who discovered the peptide corrected their original conclusion, and confirmed that this peptide originates in fact from the pig intestinal parasitic nematode, Ascaris suum. In the present study, we carried out a semi-exhaustive search for bacteria-inducible transcripts in A. suum by the cDNA subtraction method. The transcripts encoding cecropin P1 and novel Ascaris Cecropins, designated Cecropins P2, P3 and P4, were found to be positively induced factors. Chemically synthesized Ascaris Cecropins were bactericidal against a wide range of microbes, i.e. Gram-positive (Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus) and Gram-negative (Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Esherichia coli) bacteria, and were weakly but detectably active against yeasts (Saccharomyces cerevisiae and Candida albicans). Cecropin P1-like sequences were also detected at least in two other species (Ascaris lumbricoides and Toxocara canis) of the Ascarididae. All Ascaris cecropin precursors contain an acidic pro-region connected by a tetra-basic cleavage site at the C-terminus. Such an acidic pro-region is also reported to be present in the tunicate cecropin-type antimicrobial peptide styelin. On the basis of the evolutionary position of nematodes and tunicates, the ancestral cecropin may have contained the acidic pro-region at the C-terminus.

Hisashi Hirano - One of the best experts on this subject based on the ideXlab platform.

  • isolation and structure of Cecropins inducible antibacterial peptides from the silkworm bombyx mori
    Comparative Biochemistry and Physiology B, 1990
    Co-Authors: Isao Morishima, Shigeyuki Suginaka, Teruo Ueno, Hisashi Hirano
    Abstract:

    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.

Hans G Boman - One of the best experts on this subject based on the ideXlab platform.

  • antibacterial peptides designed as analogs or hybrids of Cecropins and melittin
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: D Wade, Hans G Boman, David Andreu, Anita Boman, S A Mitchell, A M V Silveira, R B Merrifield
    Abstract:

    Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from Cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.

  • DESIGN, SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF CECROPIN-LIKE MODEL PEPTIDES
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: Jürgen Fink, Hans G Boman, Anita Boman, R B Merrifield
    Abstract:

    In order to investigate structure-activity relationships of Cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of Cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C18-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural Cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.

  • sequence and specificity of two antibacterial proteins involved in insect immunity
    Journal of Immunology, 2009
    Co-Authors: Hakan Steiner, Ake Engstrom, H Bennich, Dan Hultmark, Hans G Boman
    Abstract:

    Immune responses have been described for many different insect species'. However, it is generally acknowledged that insects lack lymphocytes and immunoglobulins and their immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia 1 . The expression of this multicomponent system requires de novo synthesis of RNA and proteins 2 and its broad antibacterial activity is due to at least three independent mechanisms 3 , the most well known of which is the insect lysozyme 4-7 . However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. We recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacteral activity against Escherichia coli and several other Gram-negative bacteria'. We believe that P9A and P9B play an important part in the humoral immune responses described previously 8 and that the P9 proteins represent a new class of antibacterial agents for which we propose the name Cecropins. We describe here the primary structures of Cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom 9 which lyses both bacteria and eukaryotic cells.

  • the origin of Cecropins implications from synthetic peptides derived from ribosomal protein l1
    FEBS Letters, 1999
    Co-Authors: Katrin Putsep, Staffan Normark, Hans G Boman
    Abstract:

    We recently showed that Helicobacter pylori grown on plates produce cecropin-like antibacterial peptides to which H. pylori is resistant. This antibacterial activity was traced to fragments from the N-terminus of ribosomal protein L1 (Putsep et al., Nature, April 22, 1999). The evolutionary suggestion from this finding has now been extended by the synthesis of eight peptides with sequences taken from the N-terminus of ribosomal protein L1 (RpL1) of five different species. Two peptides of different length derived from H. pylori RpL1 showed a potent antibacterial activity, while a peptide with the sequence from Escherichia coli was 20 times less active. Like Cecropins the H. pylori peptides were not cytolytic. We suggest that the Cecropins have evolved from ribosomal protein L1 of an ancestral intracellular pathogen that developed to a symbiont ending as an organelle. When the R1 gene moved into the host nucleus, a duplication provided a copy from which today Cecropins could have evolved.

  • Interaction of the Mammalian Antibacterial Peptide Cecropin P1 with Phospholipid Vesicles
    Biochemistry, 1995
    Co-Authors: Ehud Gazit, Hans G Boman, Anita Boman, Yechiel Shai
    Abstract:

    Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect Cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect Cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect Cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect Cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.