PurposeTo illustrate decision points encountered when using evidence-based guidelines for diagnosis and management of chronic cough by means of a composite case study based on primary care practice.Data sourceThe American College of Chest Physicians (ACCP) Evidence-Based Clinical Practice Guidelines for Diagnosis and Management of Cough, and review of the scientific literature on cough and related topics.ConclusionsThe ACCP guidelines offer a systematic approach that uses trials of empirical therapies to diagnose and resolve the very common, often perplexing complaint of chronic cough. The major diagnoses reached are upper airway cough syndrome, cough-variant asthma, gastroesophageal reflux disease, and nonasthmatic eosinophilic bronchitis.Implications for practiceCough is the most common reason that patients seek an office visit. Many coughs resolve spontaneously, but coughs that persist significantly impair the quality of life. Use of the ACCP guidelines allows a stepwise empirical approach to the problem of unexplained chronic cough. This approach greatly increases the percentage of chronic coughs that are accurately diagnosed and effectively treated, and avoids unnecessary diagnostic testing.

ABSTRACT Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [ 55 Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1 - lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved, in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significant;ly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Fibrinogen gamma-chain C-terminal peptide HHLG-GAKQAGDV (gamma 12) and alpha-chain peptide GRGDSP are known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet integrin receptor GPIIb/IIIa. NMR studies of gamma 12 in the presence of purified GPIIb/IIIa in SDS/water solution have demonstrated the presence of two gamma 12 binding states, one of which is eliminated by GRGDSP (RGD) up to a RGD:gamma 12 ratio of 2:1. RGD:gamma 12 ratios greater than 2:1 produce multiple sets of gamma 12 NMR signals in TOCSY spectra. At a ratio of 4:1, two to four such resonance sets can be resolved for A405, Q407, A408, G409, D410 and V411 spin systems. The number of multiple resonances remains unchanged at ratios of 6:1 and 8:1. Addition of gamma 12 to reverse the ratio to 8:8 (1:1) has no apparent effect on the RGD-induced distribution. Results suggest that RGD irreversibly induces a conformational transition(s) in GPIIb/IIIa to produce multiple gamma 12 binding sites on the receptor.

The C-terminal dodecapeptide from human fibrinogen gamma-chain, residues 400-411, HHLG-GAKQAGDV (gamma12), is known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet glycoprotein integrin receptor GPIIb/IIIa. NMR studies of gamma12 in the presence of purified GPIIb/IIIa (230 kDa) demonstrate that two gamma12 binding states (gamma12-I and gamma12-II) are present on the integrin receptor. The N-terminal sequence HHLG is crucial to formation of gamma12 state I since in a shorter gamma-chain octapeptide, GAKQAGDV, gamma12-I is not observed. Addition of the hexapeptide GRGDSP to the gamma12-receptor preparation effectively removes the gamma12-I population, suggesting either that gamma12 and GRGDSP share one binding site or that their binding sites are allosterically linked. Distance geometry calculations using transfer NOEs from gamma12-I (gamma12-II shows practically no NOEs) indicate the presence of helix conformation when bound to the receptor. Line broadening and chemical shift changes relative to free gamma12 suggest that gamma12 interacts with GPIIb/IIIa primarily through N-terminal residues H400 to Q407.

The tripeptide RGD is well known for its role in integrin receptor-mediated cell-cell surface adhesion. Here, NMR and transferred NOE studies have been done with the fibrinogen/fibronectin-derived hexapeptide GRGDSP in the presence of sodium dodecyl sulfate (SDS) and purified platelet glycoprotein integrin receptor GPIIb/IIIa. In the presence of SDS and absence of receptor, GRGDSP gives NOE-based distance geometry-generated structures characteristic of two 'nested' beta-turns centered at RG and GD. In the presence of integrin GPIIb/IIIa, GRGDSP resonances are chemically shifted and broadened consistent with a dynamic equilibrium between free and receptor 'bound' peptide. NOEs characteristic of the nested beta-turns are either absent or weaker indicating a significant conformational change in GRGDSP in the receptor bound state. GRGDSP appears to bind the receptor in a more extended backbone conformation which positions aspartic acid and arginine residues spatially close for potential electrostatic interactions.