Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae.

In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Gram-positive bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI-TOF and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization.

Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli

The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95 percent purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli.

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray.

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.

Expression of CD95 and CD95L on astrocytes in the CA1 area of the immature rat hippocampus after hypoxia-ischemia injury.

The immature brain is affected profoundly by hypoxia-ischemia (HI) injury, which can lead to permanent neurologic sequelae in survivors. Neuronal degeneration after HI injury usually is achieved through apoptosis. Both CD95 and its natural ligand, CD95L, which are key molecules in the regulation of apoptosis, are constitutively expressed by neurons and astrocytes during embryonic and early postnatal stages. Further, CD95 or CD95L may have a functional relationship in glial cells and lead to apoptosis of these cells. The hippocampus, especially the CA1 area, is particularly susceptible to HI injury. We therefore investigated the temporal and spatial alterations in CD95 and CD95L expression in the CA1 area of 7-d-old rats after unilateral ligation of the carotid artery. Using immunohistochemistry and Western blotting, we showed that expression of CD95 and CD95L in the hippocampus peaked at 12 h and then decreased. In addition, we used terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling to demonstrate apoptosis among CD95- and CD95L-reactive cells. Our findings show that increases in the expression of CD95 and CD95L after HI injury may involve astrocytic apoptosis in the 7-d-old rat hippocampus, and these molecules may act as targets or inducers of cell death.

Characterization and cDNA cloning of hinnavin II, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae.

Hinnavins, together with lysozymes, are the main types of antibacterial peptides/proteins previously isolated from the larval haemolymph of the cabbage butterfly, Artogeia rapae as part of the humoral immune response to a bacterial invasion. One of these antibacterial peptides, named hinnavin II, was purified and characterized after cDNA cloning. The purified hinnavin II was more active against Gram negative than against Gram positive bacteria. Hinnavin II also showed a powerful synergistic effect on the inhibition of bacterial growth with purified lysozyme. The cDNA has a total length of 186 bp with a 114 coding region. The deduced protein sequence contains 38 amino acids with a coding capacity of 4142.8 Da. The result of a multiple sequence alignment and phylogenetic analysis with Clustal W indicated that mature hinnavin II showed an approximately 78.9% amino acid sequence identity with cecropin A and originated from a group containing mostly lepidopteran cecropins.

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment.

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.

Characterization of a novel repetitive secretory protein specifically expressed in the modified salivary gland of Hydropsyche sp. (Trichoptera; Hydropsychidae).

Here, we report the cloning and characterization of a common salivary gland-specific gene, Nf-1, from late instar Hydropsyche sp. larvae, and show that the corresponding gene product is translated in the gland and secreted to the gland lumen. The deduced Nf-1 protein is primarily composed of five repetitive sequence units of 63-65 amino acids, and contains a putative signal sequence composed of 19 amino acids. Secreted Nf-1 (approximately 37 kDa) was localized to the gland lumen by Western blotting of gland and lumen fractions. Together, the structure, expression pattern and protein localization of Nf-1 indicate that this protein is likely to be a major component of the silk shields and nets produced by the aquatic insect, Trichoptera.