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ABSTRACT: Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (∼14-kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has α-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1-4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained α-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.
Insect biochemistry and molecular biology 04/2013; · 3.25 Impact Factor
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Eleonora Franzetti,
Zhi-Jun Huang,
Yan-Xia Shi,
Kun Xie,
Xiao-Juan Deng,
Jian-Ping Li,
Qing-Rong Li, Wan-Ying Yang,
Wen-Nian Zeng,
Morena Casartelli,
Hui-Min Deng,
Silvia Cappellozza,
Annalisa Grimaldi,
Qingyou Xia,
Qili Feng,
Yang Cao,
Gianluca Tettamanti
Apoptosis 02/2012; 17(2):217. · 4.07 Impact Factor
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Eleonora Franzetti,
Zhi-Jun Huang,
Yan-Xia Shi,
Kun Xie,
Xiao-Juan Deng,
Jian-Ping Li,
Qing-Rong Li, Wan-Ying Yang,
Wen-Nian Zeng,
Morena Casartelli,
Hui-Min Deng,
Silvia Cappellozza,
Annalisa Grimaldi,
Qingyou Xia,
Qili Feng,
Yang Cao,
Gianluca Tettamanti
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ABSTRACT: Although several features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval-adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells.
Apoptosis 11/2011; 17(3):305-24. · 4.07 Impact Factor
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Xiang-Li Dang,
Jin-Huan Tian, Wan-Ying Yang,
Wen-Xian Wang,
Jun Ishibashi,
Ai Asaoka,
Hui-Yu Yi,
Yi-Feng Li,
Yang Cao,
Minoru Yamakawa,
Shuo-Yang Wen
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ABSTRACT: A novel antimicrobial peptide, Bactrocerin-1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin-1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin-1 showed very high similarity to the active fragment (46V-65S-NH(2)) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin-1 is a hydrophobic, positively charged, and Lys/Ile/Gly-rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin-1 showed a very broad spectrum of anti-microbial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Bactrocerin-1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 microM. Analysis of the Helical-wheel projection and the CD spectrum suggested that Bactrocerin-1 contains the amphipathic alpha-helix.
Archives of Insect Biochemistry and Physiology 06/2009; 71(3):117-29. · 1.36 Impact Factor
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ABSTRACT: Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated.
Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae.
Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.
BMC Molecular Biology 02/2009; 10:50. · 2.86 Impact Factor
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ABSTRACT: Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5' rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-kappaB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-kappaB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.
Journal of Biomedicine and Biotechnology 01/2009; 2009:315423. · 2.44 Impact Factor
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ABSTRACT: Drosomycin (Drs) gene encodes a 44-residue inducible antifungal peptide, Drosomycin, in Drosophila melanogaster. Six genes, Drs-lC, Drs-lD, Drs-lE, Drs-lF, Drs-lG and Drs-lI, show homology to the Drs form in a multigene family on the 3rd chromosome of D. melanogaster. It is the first experimental demonstration that the six members in the Drs family act as functional genes. To further delineate the functional divergence of these six members, their cDNA sequences were cloned respectively into the pET-3C vector and expressed in the E. coli. The antifungal activity of the expression products was assayed using the Cerletti's method. The results showed a difference among the six isoforms in antifungal activity against the tested fungal strains: in which Drs was most effective and showed antifungal activity to all seven fungal strains, whereas isoform Drs-lC was effective to six strains, Drs-lD was effective to five strains, Drs-lG was effective to four strains, and Drs-lE and Drs-lF were effective to only three strains. Drs-lI had no activity against any tested fungal strains. By comparing the variable residue sites of these six isoforms to that of Drosomycin in the three-dimensional structure, we suggested that the reduction in the antifungal activity was due to the variable residues that were not in the alpha-helix. In addition, two inserted residues (RV) in Drs-lI may affect the dimensional structure and resulted in a functional change. These results may explain the evolution of the Drosomycin multigene family and its functional divergence.
Gene 10/2006; 379:26-32. · 2.34 Impact Factor
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ABSTRACT: The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-IC) from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-IC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-IC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-IC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.
Agricultural Sciences in China 6(10):1209-1216. · 0.45 Impact Factor
