Genetically encoded antibiotic peptides are evolutionarily ancient and widespread effector molecules of immune defence. Mammalian defensins, one subset of such peptides, have been implicated in the antimicrobial defence capacity of phagocytic leukocytes and various epithelial cells, but direct evidence of the magnitude of their in vivo effects have not been clearly demonstrated. Paneth cells, specialized epithelia of the small intestinal crypt, secrete abundant alpha-defensins and other antimicrobial polypeptides including human defensin 5 (HD-5; also known as DEFA5). Although antibiotic activity of HD-5 has been demonstrated in vitro, functional studies of HD-5 biology have been limited by the lack of in vivo models. To study the in vivo role of HD-5, we developed a transgenic mouse model using a 2.9-kilobase HD-5 minigene containing two HD-5 exons and 1.4 kilobases of 5'-flanking sequence. Here we show that HD-5 expression in these mice is specific to Paneth cells and reflects endogenous enteric defensin gene expression. The storage and processing of transgenic HD-5 also matches that observed in humans. HD-5 transgenic mice were markedly resistant to oral challenge with virulent Salmonella typhimurium. These findings provide support for a critical in vivo role of epithelial-derived defensins in mammalian host defence.
The antimicrobial peptide human alpha-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are approximately 90 450 microg per cm2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms--HD5(56-94) and HD5(63-94)--that are cleaved at the Arg55-Ala56 and Arg62-Thr63 sites, respectively. We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.
Paneth cells (PCs) were described over a century ago as granulated cells located at the base of small intestinal crypts, the 'crypts of Lieberkühn.' Various histochemical staining procedures were developed that identified PCs based on their distinctive granule-staining pattern. Early on, PCs were proposed to perform a specialized function other than absorption of digested nutrients, the predominant task of the small intestinal epithelium. Since then, many constituents of the PC granules have been biochemically characterized. The presence of various granule-associated antimicrobial substances and their release upon microbial challenge suggest that PCs function as specialized defense cells in the small intestine. Altered resistance to microbial infection in animal models with disrupted or augmented PC function provides further support for the host defense role of PCs. Other PC components suggest that PCs may also participate in the regulation of lumenal ionic composition, crypt development, digestion, and intestinal inflammation.
Two novel
low-spin complexes,
[Et4N][FeL2]·1.5H2O
(1) and
[Et4N][CoL2]·2H2O
(2) (L is a deprotonated bis-amide ligand), have been
synthesized and characterized. Four relatively longer equatorial
M−Namide bonds and two significantly shorter axial
M−Npy bonds are the noteworthy features of their X-ray
structures. EPR and (34−300 K) magnetic studies of 1
confirm its low−spin character. Cyclic voltammetric studies
reveal a highly stabilized M(III) state. For 1 a linear
correlation between the Fe(III)−Fe(II) reduction potentials
and the reciprocal of solvent dielectric constants is
obtained.
Nucleic acids have been among the first targets for antitumor drugs and antibiotics, and with the unveiling of new biological roles in regulation of gene expression, specific DNA and RNA structures have become very attractive targets, especially when the corresponding proteins are undruggable. Biophysical assays to test target structure and ligand binding stoichiometry, affinity, specificity and binding modes are part of the drug development pipeline. Mass spectrometry offers unique advantages as a biophysical method due to its ability to distinguish each stoichiometry present in a mixture. In addition, advanced mass spectrometry approaches (reactive probing, fragmentation techniques, ion mobility spectrometry, ion spectroscopy) provide more detailed information on the complexes. Here we review the fundamentals of mass spectrometry and all its particularities when studying non-covalent nucleic acid structures, and then review what has been learned thanks to mass spectrometry on nucleic acid structures, self-assemblies (e.g., duplexes or G-quadruplexes), and their complexes with ligands. 47
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