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Aron B Fisher - One of the best experts on this subject based on the ideXlab platform.
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binding sites for interAction of peroxiredoxin 6 with SurfActAnt Protein A
Biochimica et Biophysica Acta, 2016Co-Authors: Saikumari Y Krishnaiah, Haitao Li, Chandra Dodia, Elena M Sorokina, Sheldon I Feinstein, Aron B FisherAbstract:Peroxiredoxin 6 (Prdx6) is A bifunctionAl enzyme with peroxidAse And phospholipAse A2 (PLA2) Activities. This Protein pArticipAtes in the degrAdAtion And remodeling of internAlized dipAlmitoylphosphAtidylcholine (DPPC), the mAjor phospholipid component of lung SurfActAnt. We hAve shown previously thAt the PLA2 Activity of Prdx6 is inhibited by the lung SurfActAnt-AssociAted Protein cAlled SurfActAnt Protein A (SP-A) through direct Protein-Protein interAction. Docking of SPA And Prdx6 wAs modeled using the ZDOCK (zlAb.bu.edu) progrAm in order to predict moleculAr sites for binding of the two Proteins. The predicted peptide sequences were evAluAted for binding to the opposite Protein using isothermAl titrAtion cAlorimetry And circulAr dichroism meAsurement followed by determinAtion of the effect of the SP-A peptide on the PLA2 Activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix And 83DEELQTELYEIKHQIL99 in SP-A were identified As the sites for hydrophobic interAction And H+-bonding between the 2 Proteins. TreAtment of mouse endotheliAl cells with the SP-A peptide inhibited their recovery from lipid peroxidAtion AssociAted with oxidAtive stress indicAting inhibition of Prdx6 Activity by the peptide in the intAct cell.
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PAthwAy to lAmellAr bodies for SurfActAnt Protein A.
American Journal of Physiology-lung Cellular and Molecular Physiology, 2010Co-Authors: Aron B Fisher, Chandra Dodia, Peter Ruckert, Sandra R BatesAbstract:AlveolAr SurfActAnt Protein A (SP-A) is endocytosed by type II epitheliAl cells through clAthrin-dependent uptAke And tArgeted to lAmellAr bodies for resecretion. However, the mechAnism for secretion of newly synthesized SP-A, whether regulAted exocytosis of lAmellAr bodies or constitutive secretion, is unresolved. If it is the lAtter, lAmellAr body SP-A would represent endocytosed Protein. AmAntAdine, An inhibitor of clAthrin-coAted vesicle budding, wAs used to evAluAte the role of endocytosis in AccumulAtion of SP-A in lAmellAr bodies. In isolAted rAt lungs, AmAntAdine (10 mM) inhibited uptAke of endotrAcheAlly instilled 35S-lAbeled biosynthesized SurfActAnt Proteins by >80%. To study trAfficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35S]methionine, And SurfActAnt wAs isolAted from lung lAvAge fluid And lAmellAr bodies were isolAted from lung homogenAte. With control lungs, the meAn specific Activity of [35S]SP-A (disintegrAtions per minute per microgrAm of SP-A) increAsed lineArly with time of perfusion: it wAs significAntly higher in isolAted lAmellAr bodies thAn in SurfActAnt And wAs increAsed in both compArtments by 50–60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest A precursor-product relAtionship between lAmellAr body And extrAcellulAr [35S]SP-A. Specific Activities in both compArtments were unAffected by Addition of AmAntAdine (10 mM) to the lung perfusAte, indicAting thAt uptAke from the AlveolAr spAce wAs not responsible for the increAse in lAmellAr body [35S]SP-A. Thus the pAthwAy for secretion of newly synthesized SP-A is by trAnsfer from the site of synthesis to the storAge/secretory orgAnelle prior to lAmellAr body exocytosis.
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role of p63 ckAp4 in binding of SurfActAnt Protein A to type ii pneumocytes
American Journal of Physiology-lung Cellular and Molecular Physiology, 2008Co-Authors: Sandra R Bates, Kevin Yu, Sheldon I Feinstein, Altaf S Kazi, Daniel S Gonder, Aron B FisherAbstract:We hAve recently described A putAtive receptor for lung SurfActAnt Protein-A (SP-A) on rAt type II pneumocytes. The receptor, P63, is A 63-kDA type II trAnsmembrAne Protein. CoincubAtion of type II cells with P63 Antibody (Ab) reversed the inhibitory effect of SP-A on secretAgogue-stimulAted SurfActAnt secretion from type II cells. To further chArActerize SP-A interActions with P63, we expressed recombinAnt P63 Protein in EscherichiA coli And generAted Antibodies to P63. Immunogold electron microscopy confirmed endoplAsmic reticulum And plAsmA membrAne locAlizAtion of P63 in type II cells with prominent lAbeling of microvilli. Binding chArActeristics of iodinAted SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of 125I-SP-A to type II cells demonstrAted both specific (cAlcium-dependent) And nonspecific (cAlcium-independent) components. Ab to P63 Protein blocked the specific binding of 125I-SP-A to type II cells And did not chAnge the nonspecific SP-A AssociAtion. A549 cells, A pneumocyte model cell line, expressed substAntiAl levels of P63 And demonstrAted specific binding of 125I-SP-A thAt wAs inhibited by the P63 Ab. The secretAgogue (cAMP)-stimulAted increAse in cAlcium-dependent binding of SP-A to type II cells wAs blocked by the presence of P63 Ab. TrAnsfection of type II cells with smAll interfering RNA to P63 reduced P63 Protein expression, AttenuAted P63-specific SP-A binding, And reversed the Ability of SP-A to prevent SurfActAnt secretion from the cells. Our results further substAntiAte the role of P63 As An SP-A receptor Protein locAlized on the surfAce of lung type II cells.
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identificAtion And chArActerizAtion of p63 ckAp4 ergic 63 climp 63 A SurfActAnt Protein A binding Protein on type ii pneumocytes
American Journal of Physiology-lung Cellular and Molecular Physiology, 2006Co-Authors: Nisha Gupta, Yefim Manevich, Aron B Fisher, Altaf S Kazi, Sandra R BatesAbstract:SurfActAnt Protein A (SP-A) binds to AlveolAr type II cells through A specific high-Affinity cell membrAne receptor, Although the moleculAr nAture of this receptor is uncleAr. In the present study,...
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interAction of SurfActAnt Protein A with peroxiredoxin 6 regulAtes phospholipAse A2 Activity
Journal of Biological Chemistry, 2006Co-Authors: Yongzheng Wu, Yefim Manevich, Kevin Yu, James L. Baldwin, Chandra Dodia, Sheldon I Feinstein, Aron B FisherAbstract:AbstrAct Peroxiredoxin 6 (Prdx6) is A “moonlighting” Protein with both GSH peroxidAse And phospholipAse A2 (PLA2) Activities. This Protein is responsible for degrAdAtion of internAlized dipAlmitoylphosphAtidylcholine, the mAjor phospholipid component of lung SurfActAnt. The PLA2 Activity is inhibited by SurfActAnt Protein A (SP-A). We postulAte thAt SP-A regulAtes the PLA2 Activity of Prdx6 through direct Protein-Protein interAction. RecombinAnt humAn Prdx6 And SP-A isolAted from humAn AlveolAr Proteinosis fluid were studied. MeAsurement of kinetic constAnts At pH 4.0 (mAximAl PLA2 Activity) showed Km0.35 mm And VmAx 138 nmol/min/mg of Protein. SP-A inhibited PLA2 Activity non-competitively with Ki 10 μg/ml And wAs CA2+ -independent. Activity At pH 7.4 wAs ∼50% less, And inhibition by SP-A wAs pArtiAlly dependent on CA2+. InterAction of SP-A And Prdx6 At pH 7.4 wAs shown by Prdx6-mediAted inhibition of SP-A binding to AgArose beAds, A pull-down AssAy using His-tAgged Prdx6 And Ni2 -chelAting beAds, co-immunoprecipitAtion from lung epitheliAl cells And from A binAry mixture of the two Proteins, binding After treAtment with A trifunctionAl cross-linker, And size-exclusion chromAtogrAphy. AnAlysis by stAtic light scAttering And surfAce plAsmon resonAnce showed cAlcium-independent SP-A binding to Prdx6 At pH 4.0 And pArtiAl CA2+ dependence of binding At pH 7.4. These results indicAte A direct interAction between SP-A And Prdx6, which provides A mechAnism for regulAtion of the PLA2 Activity of Prdx6 by SP-A.
Sandra R Bates - One of the best experts on this subject based on the ideXlab platform.
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PAthwAy to lAmellAr bodies for SurfActAnt Protein A.
American Journal of Physiology-lung Cellular and Molecular Physiology, 2010Co-Authors: Aron B Fisher, Chandra Dodia, Peter Ruckert, Sandra R BatesAbstract:AlveolAr SurfActAnt Protein A (SP-A) is endocytosed by type II epitheliAl cells through clAthrin-dependent uptAke And tArgeted to lAmellAr bodies for resecretion. However, the mechAnism for secretion of newly synthesized SP-A, whether regulAted exocytosis of lAmellAr bodies or constitutive secretion, is unresolved. If it is the lAtter, lAmellAr body SP-A would represent endocytosed Protein. AmAntAdine, An inhibitor of clAthrin-coAted vesicle budding, wAs used to evAluAte the role of endocytosis in AccumulAtion of SP-A in lAmellAr bodies. In isolAted rAt lungs, AmAntAdine (10 mM) inhibited uptAke of endotrAcheAlly instilled 35S-lAbeled biosynthesized SurfActAnt Proteins by >80%. To study trAfficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35S]methionine, And SurfActAnt wAs isolAted from lung lAvAge fluid And lAmellAr bodies were isolAted from lung homogenAte. With control lungs, the meAn specific Activity of [35S]SP-A (disintegrAtions per minute per microgrAm of SP-A) increAsed lineArly with time of perfusion: it wAs significAntly higher in isolAted lAmellAr bodies thAn in SurfActAnt And wAs increAsed in both compArtments by 50–60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest A precursor-product relAtionship between lAmellAr body And extrAcellulAr [35S]SP-A. Specific Activities in both compArtments were unAffected by Addition of AmAntAdine (10 mM) to the lung perfusAte, indicAting thAt uptAke from the AlveolAr spAce wAs not responsible for the increAse in lAmellAr body [35S]SP-A. Thus the pAthwAy for secretion of newly synthesized SP-A is by trAnsfer from the site of synthesis to the storAge/secretory orgAnelle prior to lAmellAr body exocytosis.
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role of p63 ckAp4 in binding of SurfActAnt Protein A to type ii pneumocytes
American Journal of Physiology-lung Cellular and Molecular Physiology, 2008Co-Authors: Sandra R Bates, Kevin Yu, Sheldon I Feinstein, Altaf S Kazi, Daniel S Gonder, Aron B FisherAbstract:We hAve recently described A putAtive receptor for lung SurfActAnt Protein-A (SP-A) on rAt type II pneumocytes. The receptor, P63, is A 63-kDA type II trAnsmembrAne Protein. CoincubAtion of type II cells with P63 Antibody (Ab) reversed the inhibitory effect of SP-A on secretAgogue-stimulAted SurfActAnt secretion from type II cells. To further chArActerize SP-A interActions with P63, we expressed recombinAnt P63 Protein in EscherichiA coli And generAted Antibodies to P63. Immunogold electron microscopy confirmed endoplAsmic reticulum And plAsmA membrAne locAlizAtion of P63 in type II cells with prominent lAbeling of microvilli. Binding chArActeristics of iodinAted SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of 125I-SP-A to type II cells demonstrAted both specific (cAlcium-dependent) And nonspecific (cAlcium-independent) components. Ab to P63 Protein blocked the specific binding of 125I-SP-A to type II cells And did not chAnge the nonspecific SP-A AssociAtion. A549 cells, A pneumocyte model cell line, expressed substAntiAl levels of P63 And demonstrAted specific binding of 125I-SP-A thAt wAs inhibited by the P63 Ab. The secretAgogue (cAMP)-stimulAted increAse in cAlcium-dependent binding of SP-A to type II cells wAs blocked by the presence of P63 Ab. TrAnsfection of type II cells with smAll interfering RNA to P63 reduced P63 Protein expression, AttenuAted P63-specific SP-A binding, And reversed the Ability of SP-A to prevent SurfActAnt secretion from the cells. Our results further substAntiAte the role of P63 As An SP-A receptor Protein locAlized on the surfAce of lung type II cells.
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identificAtion And chArActerizAtion of p63 ckAp4 ergic 63 climp 63 A SurfActAnt Protein A binding Protein on type ii pneumocytes
American Journal of Physiology-lung Cellular and Molecular Physiology, 2006Co-Authors: Nisha Gupta, Yefim Manevich, Aron B Fisher, Altaf S Kazi, Sandra R BatesAbstract:SurfActAnt Protein A (SP-A) binds to AlveolAr type II cells through A specific high-Affinity cell membrAne receptor, Although the moleculAr nAture of this receptor is uncleAr. In the present study,...
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PAthwAys for cleArAnce of SurfActAnt Protein A from the lung
American journal of physiology. Lung cellular and molecular physiology, 2005Co-Authors: Deepika Jain, Aron B Fisher, Chandra Dodia, Sandra R BatesAbstract:UptAke And degrAdAtion of 125I-SurfActAnt Protein A (SP-A) over A 1-h period wAs studied in AlveolAr cells in culture And in isolAted perfused lungs to elucidAte the mechAnism for cleArAnce of the ...
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SurfActAnt Protein A is degrAded by AlveolAr mAcrophAges
American Journal of Physiology-Lung Cellular and Molecular Physiology, 1996Co-Authors: Sandra R Bates, A. B. FisherAbstract:The metAbolism of iodinAted lung SurfActAnt Protein A (SP-A) by AlveolAr mAcrophAges in primAry culture wAs exAmined to determine the role these cells plAy in the degrAdAtion of this SurfActAnt Protein. SP-A wAs isolAted from lung lAvAge obtAined from normAl bovines, pAtients with AlveolAr Proteinosis, And silicA-treAted rAts. SP-A (0.5 microgrAm/ml) wAs incubAted for 3 h with rAt AlveolAr mAcrophAges obtAined by lung lAvAge. Cell AssociAtion And degrAdAtion of humAn And rAt SP-A wAs three times greAter thAn thAt of bovine SP-A. During the 3-h period, 50% of totAl mAcrophAge-AssociAted SP-A wAs degrAded. DegrAdAtion wAs time-, temperAture-, And concentrAtion-dependent After A 1-h lAg period. SP-A degrAdAtion wAs intrAcellulAr, since NH4Cl inhibited degrAdAtion > 50%, And mAcrophAge-conditioned medium wAs ineffective. Tenfold more SP-A wAs degrAded by mAcrophAges thAn by type II cells isolAted After elAstAse digestion of rAt lungs. There wAs little degrAdAtion of SP-A by HeLA cells. We conclude thAt AlveolAr mAcrophAges tAke up And degrAde SP-A And thus could contribute to the cAtAbolism of SP-A in the lung.
Francis X. Mccormack - One of the best experts on this subject based on the ideXlab platform.
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elucidAtion of lipid binding sites on lung SurfActAnt Protein A using x rAy crystAllogrAphy mutAgenesis And moleculAr dynAmics simulAtions
Biochemistry, 2016Co-Authors: Boon Chong Goh, Michael J Rynkiewicz, Barbara A Seaton, Klaus Schulten, Francis X. MccormackAbstract:SurfActAnt Protein A (SP-A) is A collAgenous C-type lectin (collectin) thAt is criticAl for pulmonAry defense AgAinst inhAled microorgAnisms. BifunctionAl Avidity of SP-A for pAthogen-AssociAted moleculAr pAtterns (PAMPs) such As lipid A And for dipAlmitoylphosphAtidylcholine (DPPC), the mAjor component of SurfActAnt membrAnes lining the Air-liquid interfAce of the lung, ensures thAt the Protein is poised for first-line interActions with inhAled pAthogens. To improve our understAnding of the motifs thAt Are required for interActions with microbes And SurfActAnt structures, we explored the role of the tyrosine-rich binding surfAce on the cArbohydrAte recognition domAin of SP-A in the interAction with DPPC And lipid A using crystAllogrAphy, site-directed mutAgenesis, And moleculAr dynAmics simulAtions. CriticAl binding feAtures for DPPC binding include A three-wAlled tyrosine cAge thAt binds the choline heAdgroup through cAtion-π interActions And A positively chArged cluster thAt binds the phosphoryl group. This bAsic cluster is Also criticAl for binding of lipid A, A bActeriAl PAMP And tArget for SP-A. MoleculAr dynAmics simulAtions further predict thAt SP-A binds lipid A more tightly thAn DPPC. These results suggest thAt the differentiAl binding properties of SP-A fAvor trAnsfer of the Protein from SurfActAnt DPPC to pAthogen membrAnes contAining AppropriAte lipid PAMPs to effect key host defense functions.
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elucidAtion of lipid binding sites on lung SurfActAnt Protein A using x rAy crystAllogrAphy mutAgenesis And moleculAr dynAmics simulAtions
Biochemistry, 2016Co-Authors: Boon Chong Goh, Michael J Rynkiewicz, Barbara A Seaton, Klaus Schulten, Francis X. MccormackAbstract:SurfActAnt Protein A (SP-A) is A collAgenous C-type lectin (collectin) thAt is criticAl for pulmonAry defense AgAinst inhAled microorgAnisms. BifunctionAl Avidity of SP-A for pAthogen-AssociAted moleculAr pAtterns (PAMPs) such As lipid A And for dipAlmitoylphosphAtidylcholine (DPPC), the mAjor component of SurfActAnt membrAnes lining the Air–liquid interfAce of the lung, ensures thAt the Protein is poised for first-line interActions with inhAled pAthogens. To improve our understAnding of the motifs thAt Are required for interActions with microbes And SurfActAnt structures, we explored the role of the tyrosine-rich binding surfAce on the cArbohydrAte recognition domAin of SP-A in the interAction with DPPC And lipid A using crystAllogrAphy, site-directed mutAgenesis, And moleculAr dynAmics simulAtions. CriticAl binding feAtures for DPPC binding include A three-wAlled tyrosine cAge thAt binds the choline heAdgroup through cAtion−π interActions And A positively chArged cluster thAt binds the phosphoryl group....
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unrAveling the duAl role of SurfActAnt Protein A At Atomistic detAil
Biophysical Journal, 2015Co-Authors: Boon Chong Goh, Francis X. Mccormack, Michael J Rynkiewicz, Barbara A Seaton, Klaus SchultenAbstract:SurfActAnt Protein A (SP-A) plAys An importAnt role in pulmonAry innAte immunity by recognizing cAnonicAl pAtterns on microbiAl surfAces. It protects the lungs from infection by recognizing the lipid component (lipid A) on grAm negAtive bActeriAl surfAces, And by helping to initiAte vArious cleArAnce mechAnisms. SP-A is Also known to AggregAte dipAlmitoylphosphAtidylcholine (DPPC), the mAjor constituent of lung pulmonAry SurfActAnt, to form tubulAr myelin, A highly structured form of SurfActAnt lipids And Proteins. It is uncleAr how SP-A cAn bind to both DPPC And lipid A for two very different functions. To unrAvel the duAl role of SP-A, we performed crystAllogrAphic And mutAtionAl AnAlyses As well As All-Atom moleculAr dynAmics (MD) simulAtions. We found severAl criticAl binding feAtures for lipid binding thAt involve cAtion-pi interActions And hydrogen bonds. MD simulAtions reveAled thAt SP-A binds to lipid A more tightly thAn to DPPC. These results suggest thAt SP-A mAy trAnsfer from SurfActAnt DPPC to pAthogen membrAnes to initiAte its host defense functions.
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crystAllogrAphic complexes of SurfActAnt Protein A And cArbohydrAtes reveAl ligAnd induced conformAtionAl chAnge
Journal of Biological Chemistry, 2011Co-Authors: Feifei Shang, Francis X. Mccormack, Michael J Rynkiewicz, James F Head, Tanya Cafarella, Barbara A SeatonAbstract:SurfActAnt Protein A (SP-A), A C-type lectin, plAys An importAnt role in innAte lung host defense AgAinst inhAled pAthogens. CrystAllogrAphic SP-A·ligAnd complexes hAve not been reported to dAte, limiting AvAilAble moleculAr informAtion About SP-A interActions with microbiAl surfAce components. This study describes crystAl structures of cAlcium-dependent complexes of the C-terminAl neck And cArbohydrAte recognition domAin of SP-A with d-mAnnose, d-α-methylmAnnose, And glycerol, which represent subdomAins of glycAns on pAthogen surfAces. CompArison of these complexes with the unligAnded SP-A neck And cArbohydrAte recognition domAin reveAled An unexpected ligAnd-AssociAted conformAtionAl chAnge in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D And mAnnAn-binding lectin. The net result of the conformAtionAl chAnge is thAt the SP-A lectin site And the surrounding loop region become more compAct. The Glu-202 side chAin of unligAnded SP-A extends out into the solvent And AwAy from the cAlcium ion; however, in the complexes, the Glu-202 side chAin trAnslocAtes 12.8 Å to bind the cAlcium. The AvAilAbility of Glu-202, together with positionAl chAnges involving wAter molecules, creAtes A more fAvorAble hydrogen bonding environment for cArbohydrAte ligAnds. The Lys-203 side chAin reorients As well, extending outwArd into the solvent in the complexes, thereby opening up A smAll cAtion-friendly cAvity occupied by A sodium ion. Binding of this cAtion brings the lArge loop, which forms one wAll of the lectin site, And the AdjAcent smAll loop closer together. The Ability to undergo conformAtionAl chAnges mAy help SP-A AdApt to different ligAnd clAsses, including microbiAl glycolipids And SurfActAnt lipids.
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serum SurfActAnt Protein A is A strong predictor of eArly mortAlity in idiopAthic pulmonAry fibrosis
Chest, 2009Co-Authors: Brent W Kinder, Francis X. Mccormack, Kevin K Brown, Alma Kervitsky, Marvin I Schwarz, Talmadge E KingAbstract:BAckground Serum SurfActAnt Protein A And SP-D hAd prognostic vAlue for mortAlity in pAtients with idiopAthic pulmonAry fibrosis in prior studies before the reclAssificAtion of the idiopAthic interstitiAl pneumoniAs. We hypothesized thAt bAseline serum SP-A And SP-D concentrAtions would be independently AssociAted with mortAlity Among pAtients with biopsy-proven IPF And would improve A prediction model for mortAlity. Methods We evAluAted the AssociAtion between serum SP-A And SP-D concentrAtions And mortAlity in 82 pAtients with surgicAl lung biopsy-proven IPF. Regression models with clinicAl predictors Alone And clinicAl And biomArker predictors were used to predict mortAlity At 1 yeAr. Results After controlling for known clinicAl predictors of mortAlity, we found thAt eAch increAse of 49 ng/mL in bAseline SP-A level wAs AssociAted with A 3.3-fold increAsed risk of mortAlity in the first yeAr After presentAtion. We did not observe A stAtisticAlly significAnt AssociAtion between serum SP-D And mortAlity (Adjusted hAzArd rAtio, 2.04; p=0.053). Regression models demonstrAted A significAnt improvement in the 1-yeAr mortAlity prediction model when serum SP-A And SP-D (AreA under the receiving operAtor curve [AROC], 0.89) were Added to the clinicAl predictors Alone (AROC, 0.79; p=0.03). Conclusions IncreAsed serum SP-A level is A strong And independent predictor of eArly mortAlity Among pAtients with IPF. A prediction model contAining SP-A And SP-D wAs substAntiAlly superior to A model with clinicAl predictors Alone.
Thomas R Korfhagen - One of the best experts on this subject based on the ideXlab platform.
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trAnscriptionAl regulAtion of the murine SurfActAnt Protein A gene by b myb
Journal of Biological Chemistry, 1999Co-Authors: M D Bruno, Jeffrey A Whitsett, Gary F Ross, Thomas R KorfhagenAbstract:AbstrAct SurfActAnt Protein A (SP-A) is selectively synthesized in subsets of cells lining the respirAtory epithelium, where its expression is regulAted by vArious trAnscription fActors including thyroid trAnscription fActor-1 (TTF-1). Cell-specific trAnscription of the mouse SP-A promoter is mediAted by binding of TTF-1 At four distinct cis-Active sites locAted in the 5′-flAnking region of the gene. MutAtion of TTF-1-binding sites (TBE) 1, 3, And 4 in combinAtion mArkedly decreAsed trAnscriptionAl Activity of SP-A promoter-chlorAmphenicol AcetyltrAnsferAse constructs contAining SP-A gene sequences from −256 to +45. In contrAst, the sAme mutAtions enhAnced trAnscriptionAl Activity in constructs contAining AdditionAl 5′ SP-A sequences from −399 to +45 suggesting thAt cis-Acting elements within the region −399 to −256 influence effects of TTF-1 on SP-A promoter Activity. A consensus Myb-binding site wAs identified within the region, locAted At positions −380 to −371 in the mouse gene. MutAtion of the Myb-binding site decreAsed Activity of SP-A promoter constructs in MLE-15 cells. MLE-15 cells, A cell line expressing SP-A mRNA, Also expressed B-Myb. B-Myb bound to the MBS in the SP-A gene As Assessed by electrophoretic mobility shift AssAy. While co-trAnsfection of HeLA cells with A B-Myb expression plAsmid ActivAted the trAnsfected SP-A promoter About 3-fold, co-trAnsfection of B-myb with cyclin A And cdk-2, to enhAnce phosphorylAtion of B-Myb, increAsed trAnscriptionAl Activity of SP-A constructs ApproximAtely 20-fold. TAken together, the dAtA support ActivAtion of SP-A gene promoter Activity by B-Myb which Acts At A cis-Acting element in the SP-A gene.
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SurfActAnt Protein A enhAnces respirAtory syncytiAl virus cleArAnce in vivo
Journal of Clinical Investigation, 1999Co-Authors: Ann Marie Levine, Jeffrey A Whitsett, M D Bruno, Jodie Gwozdz, James M Stark, Thomas R KorfhagenAbstract:To determine the role of SurfActAnt Protein-A(SP-A) in AntivirAl host defense, mice lAcking SP-A (SP-A–/–) were produced by tArgeted gene inActivAtion. SP-A–/– And control mice (SP-A+/+) were infected with respirAtory syncytiAl virus (RSV) by intrAtrAcheAl instillAtion. PulmonAry infiltrAtion After infection wAs more severe in SP-A–/– thAn in SP-A+/+ mice And wAs AssociAted with increAsed RSV plAque-forming units in lung homogenAtes. PulmonAry infiltrAtion with polymorphonucleAr leukocytes wAs greAter in the SP-A–/– mice. Levels of proinflAmmAtory cytokines tumor necrosis fActor-α And interleukin-6 were enhAnced in lungs of SP-A–/– mice. After RSV infection, superoxide And hydrogen peroxide generAtion wAs deficient in mAcrophAges from SP-A–/– mice, demonstrAting A criticAl role of SP-A in oxidAnt production AssociAted with RSV infection. CoAdministrAtion of RSV with exogenous SP-A reduced virAl titers And inflAmmAtory cells in the lung of SP-A–/– mice. These findings demonstrAte thAt SP-A plAys An importAnt host defense role AgAinst RSV in vivo.
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SurfActAnt Protein A binds group b streptococcus enhAncing phAgocytosis And cleArAnce from lungs of SurfActAnt Protein A deficient mice
American Journal of Respiratory Cell and Molecular Biology, 1999Co-Authors: Ann Marie Levine, Jo Rae Wright, Jeffrey A Whitsett, Gary F Ross, M D Bruno, Kim E Kurak, Wendy T Watford, Thomas R KorfhagenAbstract:SurfActAnt Protein-A (SP-A) gene–tArgeted mice cleAr group B streptococcus (GBS) from the lungs At A slower rAte thAn wild-type mice. To determine mechAnisms by which SP-A enhAnces pulmonAry cleArAnce of GBS, the role of SP-A in binding And phAgocytosis of GBS wAs Assessed in SP-A (−/−) mice infected with GBS in the presence And Absence of exogenous SP-A. CoAdministrAtion of GBS with exogenous SP-A decreAsed GBS colony counts in lung homogenAtes of SP-A (−/−) mice. SP-A bound to GBS in A cAlcium-dependent mAnner. Although pulmonAry infiltrAtion with mAcrophAges wAs not Altered in SP-A (−/−) versus wild-type mice After GBS infection, the number of AlveolAr mAcrophAges with phAgocytosed bActeriA wAs lower in the SP-A (−/−) mice thAn in the wild-type mice. When SP-A wAs coAdministered with GBS, phAgocytosis wAs significAntly increAsed. Oxygen rAdicAl production by AlveolAr mAcrophAges from SP-A (−/−) mice infected with GBS wAs decreAsed compAred with wild-type controls And wAs increAsed when SP-A (−/−) mice ...
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SurfActAnt Protein A deficient mice Are susceptible to pseudomonAs AeruginosA infection
American Journal of Respiratory Cell and Molecular Biology, 1998Co-Authors: Ann Marie Levine, Jeffrey A Whitsett, M D Bruno, James M Stark, Kim E Kurak, Thomas R KorfhagenAbstract:To determine the role of SurfActAnt Protein-A (SP-A) in host defense, the murine SP-A locus wAs tArgeted by homologous recombinAtion to produce mice lAcking SP-A. SP-A−/− And wild-type mice were infected with mucoid PseudomonAs AeruginosA by intrAtrAcheAl instillAtion. PulmonAry bActeriAl loAds were greAter in SP-A−/− thAn in wild-type mice, with increAsed numbers of mucoid P. AeruginosA in lung homogenAtes At 6 And 24 h After infection. PulmonAry infiltrAtion with polymorphonucleAr leukocytes (PMN) wAs similAr in both groups; however, An eArlier influx of PMN into the lung occurred in the SP-A−/− mice. The number of bActeriA phAgocytosed by AlveolAr mAcrophAges wAs decreAsed in the SP-A−/− mice At 1 h After infection. Superoxide-rAdicAl generAtion by PMN wAs similAr for the SP-A−/− And wild-type mice, but nitrite levels were increAsed in SP-A−/− mice. ConcentrAtions of tumor necrosis fActor-α, interleukin-6, And mAcrophAge inflAmmAtory Protein-2 (proinflAmmAtory cytokines) were greAter in bronchoAlveolAr...
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SurfActAnt Protein A deficient mice Are susceptible to group b streptococcAl infection
Journal of Immunology, 1997Co-Authors: Ann Marie Levine, Jeffrey A Whitsett, Gary F Ross, M D Bruno, Karen M Huelsman, Thomas R KorfhagenAbstract:To determine the role of SurfActAnt Protein A (SP-A) in host defense, the murine SP-A locus wAs tArgeted by homologous recombinAtion to produce mice lAcking SP-A. SP-A -/- And control mice were infected with group B streptococcus (GBS) by intrAtrAcheAl instillAtion. PulmonAry infiltrAtion 6 And 24 h following infection wAs more severe in SP-A -/- thAn in control mice, And wAs AssociAted with increAsed numbers of GBS in lung homogenAtes. DisseminAtion of GBS to the spleen wAs observed more frequently in SP-A -/- mice. PulmonAry infiltrAtion with mAcrophAges wAs similAr in both groups; however, the number of bActeriA AssociAted with AlveolAr mAcrophAges wAs decreAsed in the SP-A-deficient mice. There wAs no detectAble compensAtory increAse in SurfActAnt Protein D, the other known pulmonAry collectin, in response to GBS instillAtion. SP-A plAys An importAnt role in vivo, enhAncing cleArAnce of GBS from the lung And inhibiting systemic disseminAtion of the orgAnism.
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A Synthetic Segment of SurfActAnt Protein A: Structure, in Vitro SurfAce Activity, And in Vivo EfficAcy
Pediatric Research, 1996Co-Authors: Frans J Walther, Remedios David-cu, Carol Leung, Roberta Bruni, José Hernández-juviel, Larry M Gordon, Alan J WaringAbstract:SurfActAnt Protein A (SP-A) is A 248-residue, wAter-soluble, lipid-AssociAting Protein found in lung SurfActAnt. AnAlysis of the Amino Acid sequence using the Eisenberg hydrophobic moment Algorithm predicts thAt the SP-A segment spAnning residues 114-144 hAs high hydrophobic moments, typicAl of lipid-AssociAting AmphipAthic domAins. The secondAry structure, in vitro surfAce Activity And in vivo lung Activity of this SP-A sequence were studied with A 31-residue synthetic peptide AnAlog(A^114-144). AnAlysis of the secondAry structure using circulAr dichroism And Fourier trAnsform infrAred spectroscopy indicAted AssociAtion with lipid dispersions And A dominAnt helicAl content. SurfAce Activity meAsurements of A^114-144 with SurfActAnt lipid dispersions And the hydrophobic SurfActAnt Proteins B And C (SP-B/C) showed thAt A^114-144 enhAnces surfAce Activity under conditions of dynAmic compression And respreAding on A LAngmuir/Wilhelmy surfAce bAlAnce. Synthetic SurfActAnt dispersions contAining A^114-144 improved lung compliAnce in spontAneously breAthing, 28-d premAture rAbbits to A greAter degree thAn SurfActAnt dispersions with synthetic SP-B/C And synthetic SurfActAnt lipids Alone. These observAtions indicAte thAt inclusion of A^114-144 mAy improve synthetic prepArAtions currently used for SurfActAnt replAcement therApy.
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A Synthetic Segment of SurfActAnt Protein A: Structure, in Vitro SurfAce Activity, And in Vivo EfficAcy
Pediatric Research, 1996Co-Authors: Frans J Walther, Remedios David-cu, Carol Leung, Roberta Bruni, José Hernández-juviel, Larry M Gordon, Alan J WaringAbstract:SurfActAnt Protein A (SP-A) is A 248-residue, wAter-soluble, lipid-AssociAting Protein found in lung SurfActAnt. AnAlysis of the Amino Acid sequence using the Eisenberg hydrophobic moment Algorithm predicts thAt the SP-A segment spAnning residues 114-144 hAs high hydrophobic moments, typicAl of lipid-AssociAting AmphipAthic domAins. The secondAry structure, in vitro surfAce Activity And in vivo lung Activity of this SP-A sequence were studied with A 31-residue synthetic peptide AnAlog(A^114-144). AnAlysis of the secondAry structure using circulAr dichroism And Fourier trAnsform infrAred spectroscopy indicAted AssociAtion with lipid dispersions And A dominAnt helicAl content. SurfAce Activity meAsurements of A^114-144 with SurfActAnt lipid dispersions And the hydrophobic SurfActAnt Proteins B And C (SP-B/C) showed thAt A^114-144 enhAnces surfAce Activity under conditions of dynAmic compression And respreAding on A LAngmuir/Wilhelmy surfAce bAlAnce. Synthetic SurfActAnt dispersions contAining A^114-144 improved lung compliAnce in spontAneously breAthing, 28-d premAture rAbbits to A greAter degree thAn SurfActAnt dispersions with synthetic SP-B/C And synthetic SurfActAnt lipids Alone. These observAtions indicAte thAt inclusion of A^114-144 mAy improve synthetic prepArAtions currently used for SurfActAnt replAcement therApy.