Theophylline revisited.

Theophylline was first isolated in 1888 and remains the most commonly used medication worldwide for the treatment of asthma. It decreases the need for asthma rescue medications by people who have asthma and is an effective steroid-sparing agent for patients who tolerate it. Recently, investigators have shown that theophylline decreases airway inflammation, accelerates eosinophil apoptosis, and decreases recruitment of lymphocytes and neutrophils to the lungs at low doses. It is classified as a phosphodiesterase (PDE) inhibitor, but its therapeutic mechanism of action remains undetermined. Theophylline should be reevaluated as a long-term medication for the treatment of asthma because of its ease of use, low cost, and recent evidence of its anti-inflammatory actions.

Hidden factors in asthma.

The importance of discovering and treating hidden factors that exacerbate asthma as specified in component 2 of the 1997 National Heart Lung and Blood Institute (NHLBI) expert panel report guidelines has been overshadowed by a disproportionate emphasis on component 3 (pharmacologic therapy). This has resulted in disease management models that consist of a two-step classification-treatment system in which little regard is given to the evaluation of underlying contributing factors. In addition to the identification of environmental allergens, an aggressive evaluation of other potential factors that may contribute to asthma is essential to optimal, efficient, and cost-effective asthma care. These factors include sinusitis, allergic rhinitis, and gastroesophageal reflux. Diagnostic testing for sinusitis and/or gastroesophageal reflux is warranted even in the absence of suggestive signs or symptoms for many patients with asthma classified in the moderate and severe ranges by NHLBI guidelines. A disease management algorithm for gastroesophageal reflux disease in the patient with asthma is proposed.

IL-10 induces transcription of the gene for mouse mast cell protease-1, a serine protease preferentially expressed in mucosal mast cells of Trichinella spiralis-infected mice.

Mouse bone marrow-derived mast cells (BMMC) obtained by culturing progenitor cells with rIL-3 express mouse mast cell protease (MMCP)-5 mRNA but not MMCP-1 mRNA or MMCP-4 mRNA. In terms of mast cell differentiation, these transcripts encode one early-expressed and two late-expressed chymases, respectively. cDNA and cRNA probes were used in RNase protection assays and RNA blot analyses to study the expression of these three homologous protease genes in cultured mast cells and in helminth-infected mice. Intestinal tissue from Trichinella spiralis-infected mice, containing high numbers of mucosal mast cells, had abundant amounts of MMCP-1 mRNA but only minimal amounts of the serosal mast cell transcript that encodes MMCP-4. Exposure of mouse BMMC to rIL-10-induced transcription of the MMCP-1 gene but not the MMCP-4 gene, and a cDNA encoding MMCP-1 was obtained from these rIL-10-treated cells. The expression of MMCP-1 mRNA in BMMC depended on the continuous exposure of these cells to rIL-10, and the level of MMCP-1 mRNA (but not MMCP-5 mRNA) was substantially higher in BMMC maintained in rIL-4 and rIL-10 than in rIL-3 and rIL-10 or in rIL-3, rIL-4, and rIL-10. Thus, whereas rIL-3 elicits transcription of early expressed genes in cultured mast cells, it suppresses the transcription of late-expressed genes. These in vitro and in vivo transcription studies also indicate that rIL-10 preferentially induces differentiation of mouse progenitor cells in a mucosal mast cell-specific lineage, and that expression of granule serine protease genes is regulated in a subclass-specific manner in mouse mucosal mast cells and serosal mast cells.

Serosal mast cells maintain their viability and promote the metabolism of cartilage proteoglycans when cocultured with chondrocytes.

OBJECTIVE: To determine the consequences of mast cell (MC)-chondrocyte interactions. METHODS: Cocultured cells were analyzed histochemically, morphologically, biochemically, and functionally. RESULTS: Cocultured MC adhered to the chondrocytes and remained viable. Chondrocytes cocultured with nonactivated MC produced more proteoglycans than did chondrocytes cultured alone, and these proteoglycans possessed an intact hyaluronic acid-binding region. In contrast, most of the proteoglycans produced by chondrocytes cocultured with activated MC were degraded. CONCLUSION: These studies indicate that a complex interaction occurs in which the nonactivated MC stimulates biosynthesis and the activated MC degrades cartilage proteoglycans.

Molecular cloning of the mouse mast cell protease-5 gene. A novel secretory granule protease expressed early in the differentiation of serosal mast cells.

cDNAs were isolated that encode mouse mast cell protease-5 (MMCP-5), an approximately 30,000 Mr serine protease stored in the secretory granules of serosal mast cells (SMC) and Kirsten sarcoma virus-immortalized mast cells. Based on the deduced amino acid sequences of these cDNAs, MMCP-5 is synthesized as a 247-amino acid preproenzyme composed of a novel 19-residue hydrophobic signal peptide, a Gly-Glu activation peptide not present in other mast cell chymases, and a 226-amino acid protein that represents the mature enzyme. MMCP-5 possesses a unique Asn residue in the substrate binding cleft at residue 176 and is highly basically charged. The MMCP-5 gene was isolated, sequenced, and found to belong to a distinct subset of chymase genes. Allelic variations of the MMCP-5 gene were also detected. MMCP-5 is expressed in bone marrow-derived mast cells (BMMC), Kirsten sarcoma virus-immortalized mast cells, and SMC, but not in gastrointestinal mucosal mast cells of helminth-infected mice. The abundant levels of MMCP-5 mRNA in immature BMMC indicate that this chymase is expressed relatively early during the differentiation of mast cells. MMCP-5 is the first chymase to be molecularly cloned from progenitor mast cells and is also the first chymase shown to be expressed preferentially in the SMC subclass.

Sarcomere matrix of striated muscle: in vivo phosphorylation of titin and nebulin in mouse diaphragm muscle.

Titin and nebulin are two major protein components of a cytoskeletal matrix that coexists with thick and thin filaments within the sarcomere of a wide range of striated muscles. Purified titin and nebulin from mouse diaphragm muscle are similar in size, in relative abundance, and in amino acid composition to analogous proteins from other mammals or avians. Phosphate analysis of these nucleic-acid-free proteins indicated that both proteins contain substantial amounts of protein-bound phosphate: about 12 mol of phosphate per mole of titin subunit and 11 mol of phosphate per mole of nebulin subunit. Incubation of intact, excised mouse diaphragm with radioactive inorganic phosphate resulted in significant incorporation of radiophosphate into titin and nebulin. The identification of titin and nebulin phosphorylation was facilitated by a simple salt fractionation and nuclease digestion procedure that effectively separated titin and nebulin from radiolabeled nucleic acids. Such in vivo phosphorylation studies indicated that approximately 2 mol of phosphate per titin subunit and 5 to 7 mol of phosphate per nebulin subunit were incorporated within 5 h of incubation. The incorporation nearly doubled when the beta-adrenergic agonist, isoproterenol, or a phosphodiesterase inhibitor, theophylline, was present in the medium. For both proteins, phosphorylation occurred mainly on serine residues. Nebulin also appears to possess a smaller number of threonine sites. Taken together, our data indicate that a small proportion (20 to 40%) of the steady-state titin phosphates are rapidly turning over. In contrast, most of the nebulin phosphates (50 to 100%) are readily exchanged. The modulation of turnover by external stimuli that increase cytosolic cAMP raises the possibility that at least a portion of the multiple phosphorylation sites of titin and nebulin may be involved in the functional regulation of the sarcomere matrix.

In vivo phosphorylation of titin and nebulin in frog skeletal muscle.

Titin and nebulin are two major protein components of the sarcomere matrix in striated muscles. Purified titin and nebulin from frog (Xenopus laevis) skeletal muscle are similar in size and in amino acid composition to their mammalian or avian counterparts. Both proteins contain substantial amounts of protein-bound phosphate: about 5 to 6 per titin subunit and 8 to 9 per nebulin subunit. Injection of radioactive inorganic phosphate into the dorsal lymph sacs of Xenopus laevis resulted in the significant incorporation of radioactivity into titin and nebulin within three days of incubation. Purified titin from in vivo labeled frog gastrocnemius muscle contains one mole of radioactive phosphoserine per mole of titin subunit. These data indicate that phosphorylation of frog titin and nebulin occurs in vivo.

The interaction of tetraiodofluorescein with creatine kinase.

The dye 2',4',5',7'-tetraiodofluorescein is a potent inhibitor of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) with an apparent competitive inhibition constant with respect to MgATP2- of 2.6 - 10(-5) M. The association of the dye with the enzyme elicited a red shift in the dye's spectrum, indicative of a binding site less polar than water. The dye binds to the enzyme with an equilibrium constant of dissociation of 1.7 - 10(-5) M. MgATP or MgADP competes for the dye-binding site. Creatine binds to creatine kinase-tetraiodofluorescein complex to form a ternary complex and further causes a blue-shift in the spectrum of the bound dye. The binding of the dye to fully active creatine kinase causes conformational change that was monitored by enzyme-bound 2-mercuri-4-nitrophenol, a conformation-dependent "reporter group".