Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme.

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.

Crystallization and preliminary crystallographic study of b0220, an 'ORFan' protein of unknown function from Escherichia coli.

Newly sequenced microbial genomes continue to reveal up to 50% functionally uncharacterized 'anonymous' genes. A significant fraction of these anonymous ORFs does not exhibit any sequence similarity to any protein in the databases and constitutes a set of unique sequences, denoted 'ORFans'. The structure determination of ORFan proteins is both of evolutionary and functional interest. Here, the first crystallization of an Escherichia coli ORFan gene product, the 157 amino-acid b0220 protein, is reported. The crystals belong to the trigonal space group P3 or P3(1), with unit-cell parameters a = b = 47.2, c = 88.4 A. There are two molecules in the asymetric unit. Frozen crystals diffract to 1.6 A resolution using synchrotron radiation. Phasing was performed using multiwavelength anomalous dispersion (MAD) on the selenomethionine-substituted b0220 protein.

Chondroitin sulphate inhibits connective tissue mast cells.

1. Mast cells derive from the bone marrow and are responsible for the development of allergic and possibly inflammatory reactions. Mast cells are stimulated by immunoglobulin E (IgE) and specific antigen, but also by a number of neuropeptides such as neurotensin (NT), somatostatin or substance P (SP), to secrete numerous pro-inflammatory molecules that include histamine, cytokines and proteolytic enzymes. 2. Chondroitin sulphate, a major constituent of connective tissues and of mast cell secretory granules, had a dose-dependent inhibitory effect on rat peritoneal mast cell release of histamine induced by the mast cell secretagogue compound 48/80 (48/80). This inhibition was stronger than that of the clinically available mast cell 'stabilizer' disodium cromoglycate (cromolyn). Inhibition by chondroitin sulphate increased with the length of preincubation and persisted after the drug was washed off, while the effect of cromolyn was limited by rapid tachyphylaxis. 3. Immunologic stimulation of histamine secretion from rat connective tissue mast cells (CTMC) was also inhibited, but this effect was weaker in umbilical cord-derived human mast cells and was absent in rat basophilic leukemia (RBL) cells which are considered homologous to mucosal mast cells (MMC). Oligo- and monosaccharides were not as effective as the polysaccharides. 4. Inhibition, documented by light and electron microscopy, involved a decrease of intracellular calcium ion levels shown by confocal microscopy and image analysis. Autoradiography at the ultrastructural level showed that chondroitin sulphate was mostly associated with plasma and perigranular membranes. 5. Chondroitin sulphate appears to be a potent mast cell inhibitor of allergic and nonimmune stimulation with potential clinical implications.

Pentosanpolysulfate inhibits mast cell histamine secretion and intracellular calcium ion levels: an alternative explanation of its beneficial effect in interstitial cystitis.

PURPOSE: Mast cells are ubiquitous cells derived from the bone marrow and are responsible for allergic reactions as they release numerous vasodilatory, nociceptive and pro-inflammatory molecules in response to immunoglobulin E (IgE) and specific antigen. Mast cell secretion is also triggered by a number of peptides, such as bradykinin and substance P, and may also be involved in the development of inflammatory responses. An example is interstitial cystitis, which is a sterile painful bladder disorder that has been associated with a defective glycosaminoglycan bladder mucosal layer and an increased number of activated mast cells. Pentosanpolysulfate is a synthetic, sulfated polysaccharide that has been approved for the treatment of interstitial cystitis on the premise that it may replenish the defective glycosaminoglycan layer. We hypothesize that pentosanpolysulfate may also have an additional or alternate action on bladder mast cells. We report that pentosanpolysulfate has a powerful dose dependent inhibitory effect on mast cell release of histamine induced by the mast cell secretagogue compound 48/80. MATERIALS AND METHODS: Inhibition of mast cell secretion was documented by light and electron microscopy and extended to stimulation by substance P or IgE and antigen. RESULTS: The inhibition was more potent than that seen with the clinically available mast cell stabilizer disodium cromoglycate (cromolyn). Maximal inhibition by pentosanpolysulfate was apparent within 1 minute, was unaffected by the length of pre-incubation and persisted after the drug was washed off. In contrast, the effect of cromolyn was limited by rapid tachyphylaxis. In addition, while cromolyn has no effect on mucosal or rat basophilic leukemia cells, pentosanpolysulfate inhibited histamine secretion from both. Confocal microscopy using a calcium indicator dye showed that pentosanpolysulfate decreased intracellular calcium ion levels. CONCLUSIONS: Pentosanpolysulfate appears to be a potent inhibitor of allergic and nonimmune mast cell stimulation, which is an alternative explanation of its benefit in interstitial cystitis.