Exploring Pleiotropic Functions of Canine ß-Defensin 103: Nasal Cavity Expression, Antimicrobial Activity, and Melanocortin Receptor Activity.

Canine ß-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a ß-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.

Comparative analysis of Xenopus tropicalis hepcidin I and hepcidin II genes.

Hepcidin is an antimicrobial peptide and an iron-regulatory hormone that is conserved in fish, amphibians, and mammalians. Here we report the genomic and biochemical characterization of two amphibian hepcidins (tHEP1 and tHEP2) from the Western clawed frog (Xenopus tropicalis). Similar to fish and mammalian hepcidins, both tHEP1 and tHEP2 genes contain three exons and two introns. The predicted mature tHEP1 and tHEP2 hepcidins are a 25 amino acid peptide and a 24 amino acid peptide, respectively. Both tHEP1 and tHEP2 are strongly expressed in the liver and kidney, with detectable expression in the heart. In addition, tHEP2 is also moderately expressed in the stomach and testis. The expression of tHEP2 (but not tHEP1) in the liver is strongly induced by iron overloading, while the expression of tHEP1 (but not tHEP2) in the liver is significantly inhibited by corticosterone. Genomic analysis of the promoter regions of these two frog hepcidin genes indicates that transcription regulation factors NF-kappaB and C/EBPbeta may be involved in hepcidin regulation by iron. Hence, X. tropicalis is a useful model for the study of molecular evolution, transcriptional regulation, and structure-activity relationships of vertebrate hepcidins.

Channel catfish hepcidin expression in infection and anemia.

Hepcidin, originally identified as a 25 amino acid antimicrobial peptide made in the liver, is a key regulator of iron balance and recycling in humans and mice. Closely related hepcidin genes and peptides have also been identified in a number of fish species and in teleosts are thought to function as endogenous antibiotics involved in host defense against infection. Here we report the transcriptional regulation of hepcidin expression by infection and anemia in the channel catfish. Changes in hepcidin expression in catfish challenged with Edwardsiella ictaluri and in fish affected by channel catfish anemia (CCA) were measured by real time quantitative PCR. Hepcidin transcript levels in the livers were increased 4, 19, and 22-fold at 4, 24, and 48h following bacterial challenge, respectively. However, augmented hepcidin expression in the intestine and olfactory sac was detected only at 48h post-infection. Hepcidin transcript levels in the livers of catfish affected by CCA were less than 14% of that present in healthy counterparts. Hepatic hepcidin transcript levels correlated significantly with serum iron concentrations (r=0.54, p<0.05) and with the percent saturation of transferrin (r=0.63, p<0.05). Similar to mammalian hepcidins, channel catfish hepcidin is an iron-responsive gene and may also play important roles in innate host defense to infection and in iron homeostasis. Mammalian hepcidins may have evolved from an antimicrobial peptide and its structure and transcriptional regulatory mechanisms have been conserved throughout vertebrate evolution.

Molecular and functional characterization of bovine beta-defensin-1.

We report the biochemical and functional properties of a novel bovine beta-defensin (bBD-1). Cloned from bovine mammary papillary duct epithelia, the bBD-1 cDNA predicts a 69 amino acid propeptide that is much more similar to human beta-defensin-1 (hBD-1) than to other bovine defensins. The bBD-1 gene contains two exons and one 8.5 kb intron. Using RT-PCR, we detected the bBD-1 transcript in the teat mucosa, kidney, vagina, ovary, oviduct, and colon. A synthetic bBD-1 peptide demonstrates potent antibacterial activity against Escherichia coli. The widespread expression of bBD-1 mRNA indicates that bBD-1 may play an important role in the bovine host defense against infections.

Caspase-1 activation and mature interleukin-1ß release are uncoupled events in monocytes.

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1ß (pro-IL-1ß) and extracellular release of mature IL-1ß from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 µg/mL), and/or nigericin (Nig, 30 µmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1ß antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1ß antibodies. RESULTS: We found that pro-IL-1ß was processed to mature IL-1ß in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1ß, but not the processing of pro-IL-1ß to IL-1ß, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1ß, but not the processing of IL-1ß, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1ß processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1ß by caspase-1 and the release of mature IL-1ß from human monocytes are distinct and separable events.

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion.

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.

Structure of the hypothetical Mycoplasma protein MPN555 suggests a chaperone function.

The crystal structure of the hypothetical protein MPN555 from Mycoplasma pneumoniae (gi|1673958) has been determined to a resolution of 2.8 Angstrom using anomalous diffraction data at the Se-peak wavelength. Structure determination revealed a mostly alpha-helical protein with a three-lobed shape. The three lobes or fingers delineate a central binding groove and additional grooves between lobes 1 and 3 and between lobes 2 and 3. For one of the molecules in the asymmetric unit, the central binding pocket was filled with a peptide from the uncleaved N-terminal affinity tag. The MPN555 structure has structural homology to two bacterial chaperone proteins: SurA and trigger factor from Escherichia coli. The structural data and the homology to other chaperone proteins suggests an involvement in protein folding as a molecular chaperone for MPN555.

Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide.

RecFOR proteins are important for DNA repair by homologous recombination in bacteria. The RecO protein from Thermus thermophilus was cloned and purified, and its binding to oligonucleotides was characterized. The protein was crystallized alone and in complex with a 14-mer oligonucleotide. Both crystal forms grow under different crystallization conditions in the same space group, P3(1)21 or P3(2)21, with almost identical unit-cell parameters. Complete data sets were collected to 2.8 and 2.5 A for RecO alone and for the RecO-oligonucleotide complex, respectively. Visual comparison of the diffraction patterns between the two crystal forms and calculation of an R(merge) of 33.9% on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide.