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Last updated: 13th August 2010

Literature
A different repertoire of Galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi.
Mak P, Zdybicka-Barabas A, Cytrynska M
Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., 30-387 Krakow, Poland.
Dev. Comp. Immunol. 2010 Oct;34 (10):1129-36
Abstract
To date, functioning of insect humoral immune response is especially well described in Diptera. The mechanisms of pathogen recognition, activation of signaling pathways and regulation of antimicrobial defense peptide expression are relatively well known. The present paper demonstrates evidence that the immune system of the Lepidoptera moth, Galleria mellonella, is also able to distinguish between different classes of microorganisms and responds to the invading pathogen accordingly. G. mellonella larvae were challenged with Gram-negative and Gram-positive bacteria as well as with yeast and filamentous fungus cells. Subsequently, 24, 48 and 72 h after immunization, the concentrations of lysozyme and six defense peptides were determined in the hemolymph by the HPLC technique. The compounds studied demonstrated variability both in the kinetics of the increase as well as in the concentrations reached. The Gram-negative bacterium and filamentous fungus were particularly effective immunogens, especially affecting the levels of lysozyme, Galleria defensin, proline-rich peptide 2 and cecropin D-like peptide.
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Gastric Cancer-Specific Protein Profile Identified Using Endoscopic Biopsy Samples via MALDI Mass Spectrometry.
Kim HK, Reyzer ML, Choi IJ, Kim CG, Kim HS, Oshima A, Chertov O, Colantonio S, Fisher RJ, Allen JL, Caprioli RM, Green JE
Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland 20892, National Cancer Center, Goyang, Gyeonggi, Republic of Korea, 410-769, Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232-8575, and Protein Chemistry Laboratory, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702.
J. Proteome Res. 2010 Aug;9 (8):4123-30
Abstract
To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as alpha-defensin-1, alpha-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease.
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Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.
Ntui VO, Thirukkumaran G, Azadi P, Khan RS, Nakamura I, Mii M
Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan. ntuival@yahoo.com
Plant Cell Rep. 2010 Sep;29(9):943-54.
Abstract
Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.
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HIV-1 exposed uninfected men who have sex with men have increased levels of salivary CC-chemokines associated with sexual behavior.
Hasselrot K, Bratt G, Duvefelt K, Hirbod T, Sandstrm E, Broliden K
Department of Medicine, Solna, Infectious Disease Unit, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. klara.hasselrot@ki.se
AIDS 2010 Jun;24 (10):1569-75
Abstract
OBJECTIVES: To determine whether soluble molecules with known anti-HIV-1 activity are increased in saliva of HIV-1 exposed uninfected individuals of discordant couples of men who have sex with men (MSM), and whether the levels of these molecules are associated with genetic polymorphisms, sexual behavior and/or HIV-1 neutralizing capacity. METHODS: Saliva and PBMC were collected from exposed uninfected individuals (n=25), and low-risk controls (n=22). Levels of CCL2, CCL3, CCL4, CCL5 and CCL11 were detected by Luminex, and SLPI, LL-37, alpha-defensins and IgA2 were detected by ELISA. Single nucleotide polymorphisms (SNPs) were investigated using mass spectrometry or PCR-sequencing. HIV-1 neutralizing activity was assessed using PBMCbased neutralization assays. Self-reported questionnaires described sexual behavior. RESULTS: Exposed uninfected individuals had significantly higher levels of salivary CCL2, CCL4, CCL5 and CCL11 as compared with controls although genetic polymorphisms within the corresponding regions were equally distributed. IgA2 was also increased in exposed uninfected individuals, whereas neither CCL3, SLPI, LL-37 nor alpha-defensins differed between exposed uninfected individuals and controls. The HIV-1 neutralizing capacity of saliva was associated with higher levels of CC-chemokines (but not SLPI, LL-37, alpha-defensins or IgA2) in both exposed uninfected individuals and controls. The increased levels of CC-chemokines were associated with a higher frequency of unprotected oral sex and/or additional casual sex partners. CONCLUSION: HIV-1 exposed uninfected MSM had higher levels of salivary CC-chemokines compared with controls, this finding associated with sexual behavior rather than with genetic polymorphisms. The increased levels of CC-chemokines associated with HIV-1 neutralizing capacity in saliva.
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High efficiency preparation of bioactive human alpha-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression.
Wang A, Su Y, Wang S, Shen M, Chen F, Chen M, Ran X, Cheng T, Wang J
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury of PLA, Third Military Medical University, Chongqing 400038, People´s Republic of China.
Appl. Microbiol. Biotechnol. 2010 Aug;87 (5):1935-42
Abstract
Human alpha-defensin 6 (HD(6)), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) peptide (mHD(6)) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD(6).
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Parasitic infection protects wasp larvae against a bacterial challenge.
Manfredini F, Beani L, Taormina M, Vannini L
Department of Evolutionary Biology, University of Siena, via Aldo Moro 2, 53100 Siena, Italy.
Microbes Infect. 2010 Sep;12(10):727-35.
Abstract
Host antibacterial defense after Strepsiptera parasitization is a complex and rather unexplored topic. The way how these parasites interact with bacteria invading into the host insect during an infection is completely unknown. In the present study we demonstrate that larvae of the paper wasp Polistes dominulus are more efficient at eliminating bacteria when they are parasitized by the strepsipteran insect Xenos vesparum. We looked at the expression levels of the antimicrobial peptide defensin and we screened for the activity of other hemolymph components by using a zone of inhibition assay. Transcription of defensin is triggered by parasitization, but also by mechanical injury (aseptic injection). Inhibitory activity in vitro against the Gram positive bacterium Staphylococcus aureus is not influenced by the presence of the parasite in the wasp or by a previous immune challenge, suggesting a constitutive power of killing this bacterium by wasp hemolymph. Our results suggest either direct involvement of the parasite or that defensin and further immune components not investigated in this paper, for example other antimicrobial peptides, could play a role in fighting off bacterial infections in Polistes.
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History of eczema herpeticum is associated with the inability to induce human beta-defensin (HBD)-2, HBD-3 and cathelicidin in the skin of patients with atopic dermatitis.
Hata TR, Kotol P, Boguniewicz M, Taylor P, Paik A, Jackson M, Nguyen M, Kabigting F, Miller J, Gerber M, Zaccaro D, Armstrong B, Dorschner R, Leung DY, Gallo RL
Division of Dermatology, Department of Medicine, University of California San Diego, 9350 Campus Point Drive, La Jolla, CA 92037, U.S.A. and VA Healthcare System, San Diego, U.S.A.
The British journal of dermatology 2010 Jun
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An antimicrobial peptide regulates tumor-associated macrophage trafficking via the chemokine receptor CCR2, a model for tumorigenesis.
Jin G, Kawsar HI, Hirsch SA, Zeng C, Jia X, Feng Z, Ghosh SK, Zheng QY, Zhou A, McIntyre TM, Weinberg A
Department of Biological Sciences, Case Western Reserve University School of Dental Medicine, Cleveland, Ohio, USA. ge.jin@case.edu
PLoS ONE 2010 ;5 (6):e10993
Abstract
BACKGROUND: Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human beta-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown. METHODOLOGY: The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out. CONCLUSIONS/FINDINGS: MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1alpha (IL-1alpha), IL-6, IL-8, CCL18, and tumor necrosis factor-alpha (TNF-alpha) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.
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A novel approach to the antimicrobial activity of maggot debridement therapy.
Andersen AS, Sandvang D, Schnorr KM, Kruse T, Neve S, Joergensen B, Karlsmark T, Krogfelt KA
Department of Microbiological Surveillance and Research, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
J. Antimicrob. Chemother. 2010 Aug;65 (8):1646-54
Abstract
OBJECTIVES: Commercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments. METHODS: cDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains. RESULTS: Lucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L. CONCLUSIONS: The TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.
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Defensin of the zebra mussel (Dreissena polymorpha): molecular structure, in vitro expression, antimicrobial activity, and potential functions.
Xu W, Faisal M
Department of Pathobiology & Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
Mol. Immunol. 2010 Jul;47 (11-12):2138-47
Abstract
A 409 bp full length defensin gene was cloned and sequenced based on an expressed sequence tag (EST) obtained from a normalized zebra mussel (Dreissena polymorpha) foot cDNA library developed in our laboratory. The D. polymorpha defensin (Dpd) gene encoded a peptide with 76 amino acid residues. The mature Dpd contains 54 amino acids with a fully functional insect defensin A domain. Homologue searching against GenBank database suggested that this Dpd was phylogenetically close to defensins from a group of insects with six conserved cysteine residues. Predicted with homology-modeling method, the three-dimensional structure of Dpd also demonstrated a significant similarity with insect defensin A. The recombinant Dpd was in vitro expressed through an Escherichia coli expression system. The antimicrobial activities of the refolded recombinant Dpd were found against the growth of Morganella sp., Plesiomonas shigelloides, Edwardsiella tarda, E. coli and Staphylococcus aureus aureus (ATCC12598) with the minimal inhibition concentration (MIC) 0.35, 0.43, 1.16, 6.46, and 30.39 microM, respectively. However, with less than 50 microM no detectable inhibition activities were observed against the other four Gram-negative bacteria (Aeromonas salmonicida salmonicida, Motile Aeromonas, Flavobacterium sp., Pseudomonas fluorescens, Shewanella putrifaciens), as well as the other Gram-positive bacteria (Bacillus megaterium ATCC14581, Carnobacterium maltaromaticum ATCC27865, Enterococcus faecalis ATCC19433, and Micrococcus leteus ATCC4698), and the yeast Saccharomyces cerevisiae. The RNA fluorescent in situ hybridization (FISH) of Dpd in the zebra mussel suggested that the Dpd was expressed in a variety of tissues, such as foot, retractor muscle, ctenidium, mantle, hemocytes, gonad, digestive gland, and intestine. By using the quantitative PCR, the expression level of Dpd in the zebra mussel foot was the highest, followed by the muscle. By comparing the amount of Dpd transcripts in the zebra mussel foot under two different byssogenesis conditions (byssogenesis and non-byssogenesis) with qPCR, we found that the higher expression levels of Dpd were always associated with the byssogenesis status from 2 to 4 days after the start of byssogenesis. Findings of this study suggest that the expression of the Dpd gene was upregulated in vivo by byssogeneis, and by hemocytes in vitro upon stimulation with microbial antigens.
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