Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
Phys Rev Lett ; 108(21): 215005, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-23003274

RESUMO

The National Ignition Facility has been used to compress deuterium-tritium to an average areal density of ~1.0±0.1 g cm(-2), which is 67% of the ignition requirement. These conditions were obtained using 192 laser beams with total energy of 1-1.6 MJ and peak power up to 420 TW to create a hohlraum drive with a shaped power profile, peaking at a soft x-ray radiation temperature of 275-300 eV. This pulse delivered a series of shocks that compressed a capsule containing cryogenic deuterium-tritium to a radius of 25-35 µm. Neutron images of the implosion were used to estimate a fuel density of 500-800 g cm(-3).

2.
Biochim Biophys Acta ; 870(2): 267-78, 1986 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-3006781

RESUMO

A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.


Assuntos
Pulmão/análise , Proteolipídeos/isolamento & purificação , Surfactantes Pulmonares/isolamento & purificação , Acetilglucosaminidase , Aminoácidos/análise , Animais , Cães , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Colagenase Microbiana , Neuraminidase , Fragmentos de Peptídeos/análise , Proteínas Associadas a Surfactantes Pulmonares
3.
Biochim Biophys Acta ; 827(3): 260-7, 1985 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-3918569

RESUMO

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.


Assuntos
Apolipoproteínas A/análise , Pulmão/análise , Surfactantes Pulmonares/análise , Animais , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Focalização Isoelétrica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microscopia de Fluorescência , Peso Molecular , Ratos , Ratos Endogâmicos , Tripsina/metabolismo
4.
Biochim Biophys Acta ; 911(3): 294-305, 1987 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-3101739

RESUMO

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.


Assuntos
Glicoproteínas/análise , Proteinose Alveolar Pulmonar/metabolismo , Surfactantes Pulmonares/análise , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida , Glicosídeo Hidrolases/metabolismo , Humanos , Focalização Isoelétrica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular
5.
Hum Gene Ther ; 6(1): 31-40, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7703285

RESUMO

Surfactant protein A (SP-A) was modified by covalent linkage with polylysine of average M(r) 21 kD ([Lys]21kD-SP-A) and utilized to transfect human airway epithelial cells (H441) in vitro. Transfection of H441 cells was more efficient with [Lys]21kD-SP-A than with polylysine-DNA or unmodified SP-A-DNA complexes. Transfection with [Lys]21kD-SP-A was effective at a protein-to-DNA molar ratio of 400:1 and in the presence of an exogenous surfactant preparation, Survanta. Transfection with [Lys]21kD-SP-A was reduced in the presence of unmodified SP-A consistent with the concept of a receptor mediated uptake of protein-DNA complexes. Increased transfection efficiency correlated with decreasing diameter of the [Lys]21kD-SP-A-DNA complexes, and these complexes bound to the cell surface and pseudopodia of H441 cells. Transfection was enhanced by co-incubation with replication-deficient adenovirus. Cotransfection by [Lys]21kD-SP-A-DNA and [Lys]10kD-SP-B resulted in an additive level of reporter gene (CAT) expression. [Lys]21kD-SP-A-DNA is likely to be useful as a component of a surfactant-based DNA delivery system for transfection of airway cells.


Assuntos
Produtos Biológicos , Pulmão/citologia , Polilisina , Proteolipídeos , Surfactantes Pulmonares , Transfecção , Adenoviridae/genética , DNA/genética , DNA/ultraestrutura , Microscopia Eletrônica , Polilisina/química , Proteolipídeos/química , Proteolipídeos/ultraestrutura , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/ultraestrutura , Células Tumorais Cultivadas
6.
J Histochem Cytochem ; 40(5): 643-9, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1573247

RESUMO

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.


Assuntos
Lectinas/metabolismo , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Cães , Eletroforese em Gel de Poliacrilamida , Peroxidase do Rábano Silvestre/metabolismo , Imuno-Histoquímica , Masculino , Manose/metabolismo , Alvéolos Pulmonares/metabolismo , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Ratos , Ratos Endogâmicos
7.
J Appl Physiol (1985) ; 63(2): 692-8, 1987 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3654429

RESUMO

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.


Assuntos
Pulmão/metabolismo , Fosfatidilcolinas/metabolismo , Proteolipídeos/farmacologia , Proteína A Associada a Surfactante Pulmonar/análogos & derivados , Surfactantes Pulmonares/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células/classificação , Células Cultivadas , Fenômenos Químicos , Química , Células Epiteliais , Epitélio/metabolismo , Pulmão/citologia , Masculino , Fosfatidilcolinas/antagonistas & inibidores , Proteínas Associadas a Surfactantes Pulmonares , Ratos
8.
J Biol Chem ; 261(30): 14283-91, 1986 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-3021734

RESUMO

Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.


Assuntos
Fosfolipídeos/metabolismo , Proteolipídeos/análise , Proteína A Associada a Surfactante Pulmonar/análogos & derivados , Surfactantes Pulmonares/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cães , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Técnicas de Imunoadsorção , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Colagenase Microbiana/metabolismo , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Associadas a Surfactantes Pulmonares
9.
Biochemistry ; 30(3): 858-65, 1991 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-1988071

RESUMO

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.


Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Glicoproteínas/farmacologia , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Proteolipídeos/farmacologia , Surfactantes Pulmonares/farmacologia , Aminoácidos/análise , Animais , Ditiotreitol/farmacologia , Cães , Pulmão/química , Peso Molecular , Conformação Proteica/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares
10.
Am J Physiol Lung Cell Mol Physiol ; 280(2): L279-85, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11159007

RESUMO

Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.


Assuntos
Líquido Amniótico/metabolismo , Endotoxinas/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Vias de Administração de Medicamentos , Endotoxinas/administração & dosagem , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/metabolismo , Injeções , Pulmão/embriologia , Proteolipídeos/análise , Proteolipídeos/genética , Proteolipídeos/metabolismo , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/análise , Surfactantes Pulmonares/genética , RNA Mensageiro/metabolismo , Testes de Função Respiratória
11.
Am J Physiol Lung Cell Mol Physiol ; 280(3): L503-11, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11159034

RESUMO

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.


Assuntos
Infecções por Adenoviridae/metabolismo , Pulmão/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase/biossíntese , Tirosina/análogos & derivados , Infecções por Adenoviridae/patologia , Animais , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Feminino , Imuno-Histoquímica , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II , Surfactantes Pulmonares/metabolismo , Tirosina/metabolismo
12.
J Biol Chem ; 274(39): 27523-8, 1999 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-10488087

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteolipídeos/genética , Surfactantes Pulmonares/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Consenso , Glicoproteínas/genética , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/metabolismo , Proteolipídeos/biossíntese , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Glândula Tireoide/metabolismo , Fator Nuclear 1 de Tireoide , Transfecção
13.
Am J Physiol ; 277(6): L1104-8, 1999 12.
Artigo em Inglês | MEDLINE | ID: mdl-10600879

RESUMO

The developing lung contains surfactant protein (SP) C mRNA levels comparable to term values before mature type II cells and alveolar surfactant lipids are detectable. Estimates of the amount of mature SP-C in the alveolar lavages of preterm lungs are not available. We used an antibody to a recombinant human SP-C to measure the amount of SP-C in alveolar lavages of preterm fetal rabbits, ventilated preterm rabbits, and term rabbits. The amounts of SP-C were compared with the amounts of saturated phosphatidylcholine (Sat PC). Median Sat PC amounts increased about 680-fold, and median SP-C values increased by over 5,000-fold in alveolar washes from 27 days gestation to term. There was no increase in Sat PC or SP-C with ventilation at 27 and 28 days gestation, but ventilation increased both Sat PC and SP-C at 29 days gestation. The molar percent of SP-C relative to Sat PC also increased with gestational age and with ventilation at 29 days gestation. proSP-C was abundant in a membrane fraction from lung tissue at 27 and 28 days gestation when minimal mature SP-C was detected in alveolar washes. At 29 days and at term, proSP-C decreased in membrane fractions. The preterm lung that is surfactant lipid deficient is also severely deficient in mature SP-C.


Assuntos
Proteolipídeos/análise , Alvéolos Pulmonares/química , Alvéolos Pulmonares/embriologia , Surfactantes Pulmonares/análise , Síndrome do Desconforto Respiratório do Recém-Nascido/fisiopatologia , Animais , Animais Recém-Nascidos , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Feminino , Feto/química , Idade Gestacional , Humanos , Recém-Nascido , Fosfatidilcolinas/análise , Gravidez , Coelhos , Respiração Artificial , Volume de Ventilação Pulmonar
14.
Infect Immun ; 66(9): 4229-36, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9712772

RESUMO

To determine the effects of interleukin-4 (IL-4) on bacterial clearance from the mouse lung, transgenic mice expressing IL-4 in respiratory epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice) were infected intratracheally with Pseudomonas aeruginosa. Survival of CCSP-IL-4 mice following bacterial administration was markedly improved compared with that of control mice. While bacteria proliferated in lungs of wild-type mice, a rapid reduction in the number of bacteria was observed in the IL-4 mice as early as 6 h postinfection. Similarly, intranasal administration of IL-4 enhanced bacterial clearance from the lungs of wild-type mice. While acute and chronic IL-4 increased the numbers of neutrophils in bronchoalveolar lavage fluid, bacterial infection was associated with acute neutrophilic pulmonary infiltration, and this response was similar in the presence or absence of IL-4. Local administration or expression of IL-4 in the mouse lung enhanced pulmonary clearance of P. aeruginosa in vivo and decreased mortality following infection.


Assuntos
Interleucina-4/imunologia , Infecções por Pseudomonas/imunologia , Administração Intranasal , Animais , Modelos Animais de Doenças , Glicoproteínas/metabolismo , Interleucina-1/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Transgênicos , Neutrófilos/imunologia , Proteolipídeos/metabolismo , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
15.
J Immunol ; 158(9): 4336-40, 1997 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9126996

RESUMO

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.


Assuntos
Proteolipídeos/fisiologia , Surfactantes Pulmonares/fisiologia , Streptococcus agalactiae/imunologia , Animais , Feminino , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Knockout , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Baço/microbiologia
16.
Gene Ther ; 5(9): 1244-50, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9930326

RESUMO

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.


Assuntos
Adenosina Trifosfatases , Autoantígenos/farmacologia , Proteínas de Transporte/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Desoxirribonucleases/antagonistas & inibidores , Proteínas de Drosophila , Inibidores Enzimáticos/farmacologia , Terapia Genética/métodos , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Polilisina/farmacologia , Transfecção/métodos , Células Tumorais Cultivadas , beta-Galactosidase/genética
17.
Am J Physiol Lung Cell Mol Physiol ; 278(1): L75-80, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10645893

RESUMO

Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4. 8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.


Assuntos
Interleucina-4/farmacologia , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/farmacocinética , Animais , Interleucina-4/genética , Camundongos , Camundongos Transgênicos/genética , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Pró-Fármacos/metabolismo , Proteolipídeos/farmacocinética , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/farmacocinética , RNA Mensageiro/metabolismo
18.
Proc Natl Acad Sci U S A ; 97(11): 5972-7, 2000 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-10801980

RESUMO

Targeted ablation of the surfactant protein D (SP-D) gene caused chronic inflammation, emphysema, and fibrosis in the lungs of SP-D (-/-) mice. Although lung morphology was unperturbed during the first 2 weeks of life, airspace enlargement was observed by 3 weeks and progressed with advancing age. Inflammation consisted of hypertrophic alveolar macrophages and peribronchiolar-perivascular monocytic infiltrates. These abnormalities were associated with increased activity of the matrix metalloproteinases, MMP2 and MMP9, and immunostaining for MMP9 and MMP12 in alveolar macrophages. Hydrogen peroxide production by isolated alveolar macrophages also was increased significantly (10-fold). SP-D plays a critical role in the suppression of alveolar macrophage activation, which may contribute to the pathogenesis of chronic inflammation and emphysema.


Assuntos
Glicoproteínas/fisiologia , Peróxido de Hidrogênio/metabolismo , Ativação de Macrófagos , Macrófagos Alveolares/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Metaloendopeptidases/biossíntese , Enfisema Pulmonar/metabolismo , Fibrose Pulmonar/metabolismo , Surfactantes Pulmonares/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Meios de Cultivo Condicionados , Citocinas/análise , Indução Enzimática , Glicoproteínas/deficiência , Glicoproteínas/genética , Metaloproteinase 12 da Matriz , Camundongos , Camundongos Knockout , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Proteína D Associada a Surfactante Pulmonar , Surfactantes Pulmonares/deficiência , Surfactantes Pulmonares/genética
19.
Pediatr Res ; 31(4 Pt 1): 364-71, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1373885

RESUMO

A major blood group antigenic epitope was identified on human pulmonary surfactant protein A (SP-A). MAb and polyclonal antibodies generated against purified human SP-A aggregated blood group A human erythrocytes and immunostained epithelial cells in a variety of human tissues, consistent with the tissue distribution of major blood group antigens. SP-A MAb (MAb-8) agglutinated red cells and immunostained tissues from A or AB blood groups, but did not react with cells or tissues from O or B individuals. MAb-8 immunostaining of tissue from blood group A individuals was ablated by incubation with blood group A red cells. MAb and polyclonal antibodies directed against A blood group antigens reacted strongly with purified SP-A obtained from a blood group A individual with alveolar proteinosis. MAb and polyclonal antibodies specific for B blood group antigen failed to react with SP-A from this patient or from patients who were in blood group B. Reactivity of anti-blood group MAb was lost after treatment of SP-A with endoglycosidase-F, demonstrating its reactivity with an epitope dependent on the asparagine-linked oligosaccharide at asparagine 187. Reactivity of MAb-8 with SP-A persisted after endoglycosidase-F treatment, but was lost after digestion with collagenase as assessed by Western blot after SDS-PAGE. Reactivity of MAb to SP-A was sensitive to beta-elimination, supporting the presence of another blood group antigenic site distinct from the epitope dependent on the asparagine-linked carbohydrate. The finding that the SP-A molecule contains a major blood group epitope has implication for the clinical use of surfactant replacement preparations and diagnostic reagents based on this protein.


Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Proteolipídeos/imunologia , Surfactantes Pulmonares/imunologia , Anticorpos Monoclonais , Epitopos/imunologia , Eritrócitos/imunologia , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Proteolipídeos/isolamento & purificação , Proteolipídeos/metabolismo , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/isolamento & purificação , Surfactantes Pulmonares/metabolismo , Distribuição Tecidual
20.
Proc Natl Acad Sci U S A ; 98(11): 6366-71, 2001 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-11344267

RESUMO

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (-/-) mice were produced. The SP-C (-/-) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (-/-) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (-/-) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (-/-) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (-/-) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (-/-) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.


Assuntos
Pulmão/fisiologia , Proteolipídeos/fisiologia , Surfactantes Pulmonares/fisiologia , Animais , Lavagem Broncoalveolar , Glicoproteínas/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Medidas de Volume Pulmonar , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilcolinas/metabolismo , Proteolipídeos/genética , Proteolipídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA