We report the isolation and characterization of a novel peptide with significant sequence homology to 0-defensins from human blood filtrate. The human 0-defensin-I (hBD-I) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide, hBD-1 shares the nine conserved amino acids characteristic for ~B-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes, hBD-1 is present in nanomolar concentration in human plasma.
ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3′,4′,5‐tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB‐kanR‐EFGH operon all showed wild‐type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild‐type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid‐derived H. pylori ureI restored wild‐type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy‐ethylene‐8‐laurylether; C12E8), suggested a membrane‐limited access of urea to internal urease at neutral pH. Measurement of 14C‐urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI‐independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.
Mechanisms involved in maintaining cytoplasmic metal ion homeostasis play a central role in the adaptation of Helicobacter pylori to the changing gastric environment. An investigation of the global regulatory responses to copper ions by using RNA profiling with a threshold factor of 4.0 revealed that copper induces transcription of 19 H. pylori genes and that only the ferritin gene pfr is repressed. The 57-fold copper induction identified the HP1326 gene encoding an H. pylori-specific protein as a candidate for a novel copper resistance determinant. The HP1326 gene is expressed as a monocistronic unit, and two small HP1326 mRNAs are copper induced. The HP1326 protein is secreted and is required for copper resistance maintained by cytoplasmic copper homeostasis, as H. pylori HP1326 mutants were copper sensitive and displayed increased copper induction of HP1326 transcription as well as elevated copper repression of ferritin synthesis. The clear copper-sensitive phenotype displayed by H. pylori HP1327 and HP1328 mutants provides strong evidence that the HP1326 protein, together with the signal peptide site of the H. pylori-specific protein HP1327, whose gene is located downstream from that encoding HP1326, and the CzcB and CzcA metal efflux system component homologs HP1328 and HP1329, constitutes a novel type of copper efflux pump, as discussed below. The HP1329 gene could not be inactivated, but the 14-fold transcriptional copper induction determined by RNA profiling points towards a function of the encoded CzcA homolog in copper resistance. In summary, results from RNA profiling identified the novel H. pylori-specific copper resistance determinants CrdA (HP1326) and CrdB (HP1327), which are required for adaptation to copper-rich environmental conditions
Human hemofiltrate (HF) was evaluated regarding its content of free amino acids, proteins, and regulatory peptides. Human HF was obtained from patients with end stage renal disease (ESRD). In contrast to plasma it mainly contains low and middle weight molecules < or = 45 kDa. The content of free amino acids, peptides, and proteins in pooled filtrate was determined by amino acid analysis using ortho-phthaldialdehyde/fluorenyl methyl chloroformate (OPA/FMOC) precolumn derivatization. The total amount of peptides and proteins in human HF is 49.4 mg/L (n = 8). The levels of all free amino acids (230 mg/L) and the concentration of some regulatory peptides like insulin, endothelin, gastrin, vasopressin and angiotensin II were similar compared with blood plasma. The amount of peptides and proteins detected in the filtrate was around 0.07% of total plasma proteins, and consisted mainly of smaller proteins and peptides as shown by size exclusion chromatography (SEC). The presence of large proteins in plasma is reduced by a factor of 1500 after filtration. We conclude that human hemofiltrate is a valuable source for the large-scale extraction of regulatory peptides.
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