Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier.

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.

In vivo degradation of human fibrinogen A alpha: detection of cleavage sites and release of antithrombotic peptides.

Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide extracts from human blood filtrate, a large number of fibrinogen A alpha fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen A alpha efficiently inhibits fibrinogen binding and platelet aggregation (IC50:20-50 microM) in vitro. We conclude that in vivo degradation of fibrinogen A alpha results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation.

hBD-1: a novel beta-defensin from human plasma.

We report the isolation and characterization of a novel peptide with significant sequence homology to beta-defensins from human blood filtrate. The human beta-defensin-1 (hBD-1) 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 beta-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD-1 is present in nanomolar concentration in human plasma.

Human hemofiltrate as a source of circulating bioactive peptides: determination of amino acids, peptides and proteins.

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.

A novel 77-residue peptide from porcine brain contains a leucine-zipper motif and is recognized by an antiserum to delta-sleep-inducing peptide.

In 1977 a nonapeptide, called delta-sleep-inducing peptide (DSIP) was characterized in rabbit cerebral venous blood plasma during thalamic stimulation to induce sleep. Evidence for the existence of DSIP in the central nervous system and in numerous peripheral organs of various mammalian species has been obtained using immunochemical techniques. Later findings have revealed the existence of large forms of DSIP-like immunoreactivity. We decided to investigate the molecular identity of such large forms of DSIP-like immunoreactivity by direct isolation. We have purified and characterized using amino acid analysis, sequencing, mass spectrometry and radioimmunoassay a 77-residue peptide, denoted DIP (DSIP-immunoreactive peptide), from an acid extract of porcine brain. DIP is recognized by an antiserum raised against synthetic rabbit DSIP. The amino acid sequence of DIP, however, is not related to that of DSIP, but it contains a putative leucine-zipper motif, a proline/glutamic-acid-rich domain, three potential phosphorylation sites and exhibits an acetylated N-terminus. The N-terminal but not the C-terminal part of the newly isolated peptide shares clear homology with the sequence of a protein induced by transforming growth factor beta 1 and other growth factors in mouse osteoblastic cells. DIP is also structurally similar to a baculoviral protein p10. The function of DIP remains unclear but its involvement in transcriptional regulation is probable.