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We evaluated the Binax NOW rapid immunochromatographic membrane test (ICT) for detection of Streptococcus pneumoniae urinary antigen in a population-based prospective study of adults with community-acquired pneumonia (CAP). ICT was performed with urine samples obtained from 452 (91.7%) of 493 patients enrolled. Pneumococcal antigen was detected in 19 (70.4%) of 27 patients with pneumococcal pneumonia. The test results were more frequently positive for patients who had not received antibiotics before testing (26.6% vs. 12.1%; P=.002). Only 16 (10.3%) of 156 samples obtained from patients with nonpneumococcal pneumonia yielded a positive result. Of the 269 patients who had pneumonia with no pathogen identified, antigen was detected in 69 (25.7%). With conventional microbiological criteria used as the "gold standard," the test had a sensitivity of 70.4% and a specificity of 89.7%. Testing concentrated urine samples with the ICT may be a useful technique for rapid diagnosis of pneumococcal pneumonia in adults with CAP.

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Background:In the present study we analyze, in patients with breast cancer, the tumor expression of androgen receptors (AR), its relationship with clinicopathological characteristics and with the expression of several matrix metalloproteases (MMPs) and their inhibitors (TIMPs), as well as with prognosis.

When challenged by low-iron conditions several Gram-negative pathogens secrete a hemophore (HasA) to scavenge hemin from its host and deliver it to a receptor (HasR) on their outer membrane for internalization. Here we report results from studies aimed at probing the structural and dynamic processes at play in the loading of the apo-hemophore secreted by P. aeruginosa (apo-HasAp) with hemin. The structure of apo-HasAp shows a large conformational change in the loop harboring axial ligand His32 relative to the structure of holo-HasAp, whereas the loop bearing the other axial ligand, Tyr75, remains intact. To investigate the role played by the axial ligand-bearing loops in the process of hemin capture we investigated the H32A mutant, which was found to exist as a monomer in its apo-form and as a mixture of monomers and dimers in its holoform. We obtained an X-ray structure of dimeric H32A holo-HasAp, which revealed that the two subunits are linked by cofacial interactions of two hemin molecules and that the conformation of the Ala32 loop in the dimer is identical to that exhibited by the His32 loop in wild type apoHasAp. Additional data suggest that the conformation of the Ala32 loop in the dimer is mainly a consequence of dimerization. Hence, to investigate the effect of hemin loading on the topology of the His32 loop we also obtained the crystal structure of monomeric H32A holo-HasAp coordinated by imidazole (H32A-imidazole) and investigated the monomeric H32A HasAp and H32A-imidazole species in solution by NMR spectroscopy. The structure of H32A-imidazole * To whom correspondence should be addressed: mrivera@ku.edu.Coordinates and structure factors have been deposited to the Protein Databank with the accession codes 3MOK (Apo-HasAp), 3MOL (HasAp H32A dimer) and 3MOM (HasAp H32A imidazole complex). SUPPORTING INFORMATION AVAILABLEA view of apo-HasAp showing the location of Na + and PO 4 3 − ions, simulation system of hemin bound apo-HasAp with and without KCl ions or solution, apo-HasAp His32 loop region showing the stabilizing aromatic side chains, resonance Raman and EPR spectra of H32A holo-HasAp, electronic absorption spectra and binding curve of imidazole binding to H32A holo-HasAp, 15 N, 1 H-HSQC-TROSY spectra of H32A HasAp and H32A-imidazole, tables of backbone NMR assignments for H32A HasAp and H32A-imidazole, and complete references 30, 38 and 39 . This material is available free of charge via the Internet at

Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoffé, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide αβ fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human met-hemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15 Nlabeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15 N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, † This work was supported by grants from the National Institute of Health, GM-50503 (M.R.), NSF-MCB-0818488 (M.R.) and NSF-MCB-0811888 (P.M.L.). ‡ Coordinates and crystallographic structure factors for HasAp have been deposited in the protein data bank under accession code 3ELL.Backbone resonance assignments for truncated and full-length holo-HasAp have been deposited in the BMRB under access code 15962 and 15963, respectively. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.The preferred aerobic metabolism of Pseudomonas aeruginosa requires respiratory enzymes that need iron or iron-containing cofactors for their function. The extremely low concentrations of free iron in mammalian hosts trigger a stress response in the opportunistic P. aeruginosa (and in many other pathogens) that involves the deployment of several iron-acquisition systems (1-4). The systems involved in the capture of iron typically fall in two categories, (i) excretion of low-molecular weight i...

The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of (1)H and (13)C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S = (1)/(2), (d(xy))(2)(d(xz), d(yz))(3) electron configuration and planar heme and a second with a novel S = (3)/(2), (d(xz), d(yz))(3)(d(xy))(1)(d(z)(2))(1) spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the sigma-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH(-) (J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.

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