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Last updated: 13th August 2010

Two Novel Duck Antibacterial Peptides, Avian beta-Defensins 9 and 10, with Antimicrobial Activity.
Ma D, Liao W, Wang R, Han Z, Liu S
Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China.
J. Microbiol. Biotechnol. 2009 Nov;19 (11):1447-55
Two novel avian beta-defensins (AvBDs) isolated from duck liver were characterized and their homologies with other AvBDs were analyzed. They were shown to be duck AvBD9 and AvBD10. The mRNA expression of the two genes was analyzed in 17 different tissues from 1-28-dayold ducks. AvBD9 was differentially expressed in the tissues, with especially high levels of expression in liver, kidney, crop, and trachea, whereas AvBD10 was only expressed in the liver and kidney of ducks at all the ages investigated. We produced and purified GST-tagged recombinant AvBD9 and AvBD10 by expressing the two genes in Escherichia coli. Both recombinant proteins exhibited antimicrobial activity against several bacterial strains. The results revealed that both recombinant proteins retained their antimicrobial activities against Staphylococcus aureus under a range of different temperatures (-70 degrees -100 degrees ) and pH values (pH 3-12).
Treponema denticola suppresses expression of human {beta}-defensin-3 in gingival epithelial cells through inhibition of the toll-like receptor 2 axis.
Shin JE, Kim YS, Oh JE, Min BM, Choi Y
Department of Oromaxillofacial Infection & Immunity, School of Dentistry and Dental Research Institute, Seoul National University, 28-2 Yeongeon-dong, Jongno-gu, Seoul 110-749, South Korea.
Infect. Immun. 2010 Feb;78 (2):672-9
We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human beta-defensins (HBDs) in human gingival epithelial cells. To identify the mechanisms involved in this suppression, immortalized and normal human gingival epithelial cells were infected with live or heat-killed T. denticola for 24 h, and then the expression of HBDs was examined by real-time RT-PCR. Live T. denticola suppressed the expression of HBD-3 substantially and also suppressed the expression of HBD-1 and HBD-2. However, heat-killed bacteria did not produce a suppressive effect but instead slightly upregulated the levels of HBD-2 and HBD-3. In contrast to live T. denticola, which reduced the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-kappaB signaling pathways. Knockdown of Toll-like receptor 2 (TLR2) via RNA interference abolished the suppressive effect of T. denticola on the expression of HBD-3. Heat-killed T. denticola but not live bacteria could activate TLR2 in CHO/CD14/TLR2 reporter cells, suggesting that T. denticola contains a heat-labile inhibitor(s) of TLR2 in addition to ligands recognized by TLR2. Indeed, live T. denticola was able to inhibit TLR2 activation by Pam(3)CSK. In conclusion, T. denticola suppressed the expression of HBD-3 by inhibiting the TLR2 axis in gingival epithelial cells. These results may provide new insight into the pathogenesis of periodontitis caused by T. denticola.
Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment.
Arijs I, De Hertogh G, Lemaire K, Quintens R, Van Lommel L, Van Steen K, Leemans P, Cleynen I, Van Assche G, Vermeire S, Geboes K, Schuit F, Rutgeerts P
Department of Gastroenterology, University Hospital Gasthuisberg, Leuven, Belgium.
PLoS ONE 2009 ;4 (11):e7984
BACKGROUND: Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD. METHODOLOGY/PRINCIPAL FINDINGS: Mucosal gene expression of 81 AMPs was assessed in 61 IBD patients before and 4-6 weeks after their first infliximab infusion and in 12 control patients, using Affymetrix arrays. Quantitative real-time reverse-transcription PCR and immunohistochemistry were used to confirm microarray data. The dysregulation of many AMPs in colonic IBD in comparison with control colons was widely restored by infliximab therapy, and only DEFB1 expression remained significantly decreased after therapy in the colonic mucosa of IBD responders to infliximab. In ileal Crohn´s disease (CD), expression of two neuropeptides with antimicrobial activity, PYY and CHGB, was significantly decreased before therapy compared to control ileums, and ileal PYY expression remained significantly decreased after therapy in CD responders. Expression of the downregulated AMPs before and after treatment (DEFB1 and PYY) correlated with villin 1 expression, a gut epithelial cell marker, indicating that the decrease is a consequence of epithelial damage. CONCLUSIONS/SIGNIFICANCE: Our study shows that the dysregulation of AMPs in IBD mucosa is the consequence of inflammation, but may be responsible for perpetuation of inflammation due to ineffective clearance of microorganisms.
Defensin promoters as potential tools for engineering disease resistance in cereal grains.
Kovalchuk N, Li M, Wittek F, Reid N, Singh R, Shirley N, Ismagul A, Eliby S, Johnson A, Milligan AS, Hrmova M, Langridge P, Lopato S
Australian Centre for Plant Functional Genomics, Hartley Grove, Urrbrae, SA, Australia.
Plant Biotechnol. J. 2010 Jan;8 (1):47-64
Engineering of plant protection in cereals requires well characterized tissue-specific and wounding/pathogen-inducible promoters for targeted expression of pathogen responsive and resistance genes. We describe the isolation of seven wheat and rice defensin genes expressed in early developing grain and during grain germination, two developmental stages that are particularly vulnerable to pathogens and insects. Comparison of three-dimensional (3D) models of these rice and wheat PRPI defensins indicated variations in spatial architectures that could reflect their functional diversities. Wheat and rice were stably transformed with promoter-GUS fusion constructs and the spatial and temporal activities of four promoters were studied using whole-mount and histological assays. PRPI promoters were active before and at anthesis in both transgenic wheat and rice with activity mainly in the ovary. In rice, GUS activity was also observed in vascular tissue of the lemma, palea and anthers. After fertilization, GUS was strongly expressed in the outer cell layers of the pericarp and in the main vascular bundle of the grain. During, and a short time after, seed germination, wheat promoters were active in transgenic rice embryos, roots and/or coleoptiles. All wheat and rice promoters were strongly induced by wounding in leaf, stem and grain of transgenic rice plants. These results suggest that PRPI promoters will be useful for specific targeting and accumulation of proteins conferring resistance to pathogens in vulnerable tissues of developing and germinating grain.
Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity.
Dos Santos IS, Carvalho AD, de Souza-Filho GA, do Nascimento VV, Machado OL, Gomes VM
Universidade Estadual do Norte Fluminense, Laboratório de Fisiologia e Bioquímica de Microrganismos, Campos dos Goytacazes 28013-602, RJ, Brazil.
Protein Expr Purif. 2010 May;71(1):8-15.
Plant defensins make up a family of cationic antimicrobial peptides with a characteristic three-dimensional folding pattern stabilized by four disulfide bridges. The aim of this work was the purification and functional expression of a defensin from cowpea seeds and the assessment of its alpha-amylase inhibitory activity. The cDNA encoding the cowpea defensin was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform Escherichia coli cells. The recombinant peptide was purified via affinity chromatography on a Ni Sepharose column and by reverse-phase chromatography on a C2/C18 column using HPLC. N-terminal amino acid sequencing revealed that the recombinant peptide had a similar sequence to that of the defensin isolated from seeds. The natural and recombinant defensins were submitted to the alpha-amylase inhibition assay. The cowpea seed defensin was found to inhibit alpha-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. alpha-Amylase inhibition assays also showed that the recombinant defensin inhibited alpha-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian alpha-amylases. The three-dimensional structure of the recombinant defensin was modeled, and the resulting structure was found to be similar to those of other plant defensins.
Structural Characterization and Cytolytic Activity of a Potent Antimicrobial Motif in Longicin, a Defensin-Like Peptide in the Tick Haemaphysalis longicornis.
Rahman M, Tsuji N, Boldbaatar D, Battur B, Liao M, Umemiya-Shirafuji R, You M, Tanaka T, Fujisaki K
Department of Frontier Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan.
J Vet Med Sci. 2010 Feb;72(2):149-56.
Longicin, a defensin-like peptide, was recently identified in the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analogs (P4) displayed antimicrobial/fungicidal/parasiticidal activity. In the present study, we compared longicin-derived synthetic analogs in order to characterize the antimicrobial motif (P4) by analyzing some structural features using various bioinformatics tools and/or CD spectroscopy. According to the chemicophysical characteristics, P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of a beta-sheet, which was also observed in the modeled tertiary structure. CD spectroscopy results also showed the existence of a beta-sheet and transition to a helical conformation in the presence of membrane-mimicking conditions. These structural observations on P4 suggested that the antimicrobial activity could be due to the beta-sheet as well as the alpha-helix. In addition, a sequence homology search showed that molecules identified in other ticks and organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved domain. The peptide P4 also showed low cytolytic activity. Based on the present result and previously reported studies, the peptide P4 could be suggested as a novel antimicrobial domain as well as a future therapeutic agent against bacteria.
Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.
Wu M, McClellan SA, Barrett RP, Zhang Y, Hazlett LD
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
J. Immunol. 2009 Dec;183 (12):8054-60
Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine beta-defensins (mBD) 3 and 4, the murine homologs of human beta-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-gamma, MIP-2, IL-1beta, TNF-alpha, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-kappaB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.
Expression of beta-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica.
Erles K, Brownlie J
The Royal Veterinary College, Department of Pathology and Infectious Diseases, Hawkshead Lane, Hatfield AL9 7TA, United Kingdom.
Vet Immunol Immunopathol. 2010 May 15;135(1-2):12-9.
beta-Defensins are cationic peptides which form part of the innate immune response of the respiratory epithelium. Due to their antimicrobial properties and immunostimulatory activity, beta-defensins are potential tools for the treatment and prevention of respiratory disease. In dogs, infectious respiratory disease is a common problem, particularly in housed animals. This study aimed to assess the presence of four beta-defensins in the canine respiratory tract and to use quantitative real-time PCR to determine mRNA levels following microbial challenge. Three beta-defensins, CBD1, CBD103 and CBD108, were detected in respiratory cells. All three defensins were also readily expressed in skin samples, while their expression in lymphoid tissues and the kidney was low and inconsistent. Treatment of primary tracheal epithelial cells with lipopolysaccharide (LPS) or infection with canine respiratory coronavirus led to decreased expression of CBD103 and CBD108, while cells infected with canine parainfluenza virus had lower levels of CBD1 and CBD108. Furthermore CBD103 was demonstrated to have antimicrobial activity against the respiratory pathogen Bordetella bronchiseptica.
In vivo release of alpha-defensins in plasma, neutrophils and CD8 T-lymphocytes of patients with HIV infection.
D´Agostino C, Lichtner M, Mastroianni CM, Ceccarelli G, Iannetta M, Antonucci S, Vullo V, Massetti AP
Department of Infectious and Tropical Diseases, Sapienza University, Rome, Italy.
Curr. HIV Res. 2009 Nov;7 (6):650-5
alpha-Defensins are reported to be a soluble component of innate immunity actively participating in host defense against HIV. In order to further investigate the role of alpha-defensins in innate immunity during HIV infection, we analyzed CD8+ T lymphocytes and neutrophils obtained from 34 HIV-infected and 14 uninfected subjects. CD8+ T cells and neutrophils were labelled for evaluating alpha-defensin expression by flow cytometric analysis using a dual laser FACScalibur. Culture supernatants and plasma were also collected for ELISA quantification of alpha-defensins. The results showed a significantly increased production of alpha-defensins in plasma, neutrophils and CD8 T-lymphocytes of patients with HIV infection in comparison with healthy controls. The expression of alpha-defensins, by CD8+ cells probably reflects both the intrinsic production and the uptake from cocultured cells that release defensins. The upregulation of alpha-defensin expression within neutrophils could account for the increased release of such peptides in the systemic circulation. Antiretroviral treatment did not have any effect on plasma levels and expression of alpha-defensins by neutrophils. Overall, our findings suggest that the increased production/expression of alpha-defensins could be correlated with the chronic process of immune activation seen in HIV infection.
Defensin from disk abalone Haliotis discus discus: Molecular cloning, sequence characterization and immune response against bacterial infection.
De Zoysa M, Whang I, Lee Y, Lee S, Lee JS, Lee J
Department of Marine Life Sciences, College of Ocean Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
Fish Shellfish Immunol. 2010 Feb;28(2):261-266.
Gene-encoded antimicrobial peptides (AMPs) serve a major role in host defense systems against pathogens. In this study, cDNA of a new mollusk defensin was identified from a normalized cDNA library constructed from whole tissues of disk abalone. Abalone defensin peptide (pro-defensin) has a 198-bp coding sequence comprised of a putative 66 amino acids with a mature defensin consisting of 48 amino acid residues. The presence of an invertebrate defensin family domain, an arrangement of six cysteine residues and their disulfide linkage in C(1)-C(4), C(2)-C(5) and C(3)-C(6) form, an alpha helix in three-dimensional structure and a phylogenetic relationship suggests that abalone defensin could be a new member of the invertebrate defensin family, and related to arthropod defensins. In non-stimulated abalone, defensin transcripts were constitutively expressed in all examined tissues including hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas. It was observed that abalone defensin transcripts were significantly induced in hemocytes, gills and digestive tract at different time intervals after infection by pathogenic bacteria mixture containing Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes. Our overall results suggest that disk abalone defensin could be involved in the immune response reactions as a host defense against pathogenic bacteria.