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1.
Transpl Infect Dis ; 16(3): 505-10, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24725139

ABSTRACT

The optimal combination of galactomannan index (GMI) testing for the diagnosis of invasive pulmonary aspergillosis (IPA) remains unclear. For diagnostic approaches that are triggered by clinical signs and symptoms in high-risk patients, institutional variation remains, with some centers routinely relying on only serum GMI or bronchoalveolar lavage (BAL) GMI testing. In addition, use of mold-active agents before diagnosis of IPA is becoming increasingly common, and understanding the effect of these drugs on test yield is important when making time-critical treatment decisions. In a single-center cohort of 210 allogeneic hematopoietic cell transplant recipients, we found that serum and BAL GMI testing contributed independently to IPA diagnosis, supporting the practice of sending both tests simultaneously to ensure a timely diagnosis of IPA. BAL GMI sensitivity was not affected by receipt of mold-active therapy in our cohort.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , Invasive Pulmonary Aspergillosis/diagnosis , Mannans/blood , Transplantation/adverse effects , Adolescent , Adult , Aged , Aspergillus/isolation & purification , Female , Galactose/analogs & derivatives , Humans , Invasive Pulmonary Aspergillosis/microbiology , Male , Middle Aged , Retrospective Studies , Young Adult
2.
J Infect Dis ; 198(2): 176-85, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18500928

ABSTRACT

Echinocandins target fungal beta-1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of Aspergillus fumigatus, they do induce morphological changes in fungal hyphae. Because beta-1,3 glucans activate host antifungal pathways via the Dectin-1 receptor, we investigated the effect of echinocandins on inflammatory responses to A. fumigatus. Caspofungin- or micafungin-treated conidia and germlings induced less secretion of tumor necrosis factor (TNF) and CXCL2 by macrophages than did their untreated counterparts. Diminished secretion of TNF and CXCL2 correlated with diminished beta-glucan exposure on echinocandin-treated germ tubes. In contrast to treated conidia and germlings, echinocandin-treated hyphae stimulated increased release of TNF and CXCL2 by macrophages and demonstrated intense staining with a beta-glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin-induced morphological changes in A. fumigatus hyphae are accompanied by increased beta-glucan exposure, with consequent increases in Dectin-1-mediated inflammatory responses by macrophages.


Subject(s)
Antifungal Agents/therapeutic use , Aspergillosis/drug therapy , Aspergillus fumigatus/drug effects , Echinocandins/therapeutic use , Inflammation/drug therapy , Inflammation/microbiology , beta-Glucans/metabolism , Aspergillus fumigatus/growth & development , Aspergillus fumigatus/physiology , Candida albicans/drug effects , Caspofungin , Chemokine CXCL2/drug effects , Chemokine CXCL2/metabolism , Humans , Lipopeptides , Microscopy, Confocal , Tumor Necrosis Factor-alpha/metabolism
3.
J Biol Chem ; 276(31): 29091-7, 2001 Aug 03.
Article in English | MEDLINE | ID: mdl-11395481

ABSTRACT

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.


Subject(s)
Carrier Proteins/chemistry , Membrane Proteins/chemistry , Vesicular Transport Proteins , Amino Acid Sequence , Animals , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cartilage/chemistry , Extracellular Matrix Proteins/chemistry , Glutaral , Glycoproteins/chemistry , Macromolecular Substances , Matrilin Proteins , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Microscopy, Electron , Microscopy, Electron, Scanning , Molecular Sequence Data , Molecular Weight , N-Ethylmaleimide-Sensitive Proteins , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Solutions , Thrombospondin 1/chemistry
4.
Mol Cell ; 2(5): 539-48, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9844627

ABSTRACT

The structure of 20 S particles, consisting of NSF, SNAPs, and SNARE complexes, was analyzed by electron microscopy and fluorescence resonance energy transfer. Structural changes associated with the binding of alpha-SNAP and NSF to SNARE complexes define the contribution of each component to the 20 S particle structure. The synaptic SNARE complex forms a 2.5 x 15 nm rod. alpha-SNAP binds laterally to the rod, increasing its width but not its length. NSF binds to one end of the SNAP/SNARE complex; the resulting 20 S particles measure 22 nm in length and vary in width from 6 nm at their narrowest point to 13.5 nm at their widest. The transmembrane domains of VAMP and syntaxin emerge together at the NSF-distal end of 20 S particles, adjacent to the amino terminus of alpha-SNAP.


Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins , Adenosine Triphosphate/metabolism , Animals , Brain , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Carrier Proteins/ultrastructure , Centrifugation, Density Gradient , Dimerization , Energy Transfer , Escherichia coli/genetics , Fluorometry , Glutathione Transferase/metabolism , Luminescent Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Membrane Proteins/ultrastructure , Microscopy, Immunoelectron , N-Ethylmaleimide-Sensitive Proteins , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/ultrastructure , Protein Binding , Protein Conformation , Proto-Oncogene Proteins c-myc/immunology , Qa-SNARE Proteins , R-SNARE Proteins , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
5.
J Biol Chem ; 273(25): 15675-81, 1998 Jun 19.
Article in English | MEDLINE | ID: mdl-9624162

ABSTRACT

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.


Subject(s)
Adenosine Triphosphatases/chemistry , Carrier Proteins/chemistry , Models, Chemical , Vesicular Transport Proteins , Adenosine Triphosphatases/ultrastructure , Carrier Proteins/ultrastructure , Escherichia coli , Light , Microscopy, Electron, Scanning Transmission , N-Ethylmaleimide-Sensitive Proteins , Peptide Fragments/chemistry , Protein Conformation , Scattering, Radiation , Solutions
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