AB3432 Sigma-AldrichAnti-Prosurfactant Protein B Antibody, CT

We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.

Nitric oxide signaling has an important role in regulating pulmonary development and function. Expression of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI), both critical mediators of nitric oxide (NO) signaling, is diminished in the injured newborn lung through unknown mechanisms. Recent studies suggest that excessive transforming growth factor-beta (TGF-beta) activity inhibits injured newborn lung development. To explore mechanisms that regulate pulmonary NO signaling, we tested whether TGF-beta decreases sGC and PKGI expression in the injured developing lung and pulmonary vascular smooth muscle cells (SMC). We found that chronic oxygen-induced lung injury decreased pulmonary sGCalpha(1) and PKGI immunoreactivity in mouse pups and that exposure to a TGF-beta-neutralizing antibody prevented this reduction of sGC and PKGI protein expression. In addition, TGF-beta(1) decreased expression of NO signaling enzymes in freshly isolated pulmonary microvascular SMC/myofibroblasts, suggesting that TGF-beta has a direct role in modulating NO signaling in the pup lung. Moreover, TGF-beta(1) decreased sGC and PKGI expression in pulmonary artery and aortic SMC from adult rats and mice, suggesting a general role for TGF-beta in modulating NO signaling in vascular SMC. Although other cytokines decrease sGC mRNA stability, TGF-beta did not modulate sGCalpha(1) or PKGIbeta mRNA turnover in vascular SMC. These studies indicate for the first time that TGF-beta decreases NO signaling enzyme expression in the injured developing lung and pulmonary vascular SMC. Moreover, they suggest that TGF-beta-neutralizing molecules might counteract the effects of injury on NO signaling in the newborn lung.

Inability to produce surfactant protein B (SP-B) causes fatal neonatal respiratory disease. A frame-shift mutation (121ins2) is the predominant but not exclusive cause of disease. To determine the range of mechanisms responsible for SP-B deficiency, both alleles from 32 affected infants were characterized. Sixteen infants were homozygous for the 121ins2 mutation, 10 infants were heterozygous for the 121ins2 and another mutation, and six infants were homozygous for other mutations. Thirteen novel SP-B gene mutations were identified, which were not found in a control population. One novel mutation was found in two unrelated families. Surfactant protein expression was evaluated by immunohistochemistry and/or protein blotting. Absence of proSP-B and mature SP-B was associated with nonsense and frame-shift mutations. In contrast, proSP-B expression was associated with missense mutations, or mutations causing in-frame deletions or insertions, and low levels of mature SP-B expression were associated with four mutations. Extracellular staining for proSP-C and/or aberrantly processed SP-C was observed in lungs of all infants with SP-B gene mutations. Hereditary SP-B deficiency is caused by a variety of distinct mutations in the SP-B gene and may be associated with reduced, as well as absent, levels of mature SP-B, likely caused by impaired processing of proSP-B.

Although replication-deficient adenoviruses efficiently transfer genes into epithelial cells of the lung, host immune responses limit the extent and duration of gene expression. To define further the role of inflammatory responses to first-generation, recombinant, deltaE1, deltaE3 adenovirus in lung pathology and surfactant protein homeostasis, expression of the surfactant proteins SP-A, SP-B, and proSP-C was determined by immunohistochemistry 2, 7, and 14 days following intratracheal administration of 2 x 10(9) pfu of a recombinant adenovirus, Av1Luc1, to BALB/c nu/nu and BALB/c wild-type mice. Two to 7 days after virus administration, an acute inflammatory response was observed in both mouse strains. Respiratory epithelial cells were sloughed, and extracellular accumulation of SP-A and SP-B was detected in the airways. Diminished immunostaining for SP-A and SP-B was noted in type II cells, and SP-A and SP-B mRNA expression was decreased in focal regions of the lungs from both mouse strains. One week after virus administration, immunostaining for proSP-C was markedly increased in cells lining the regenerating alveolar epithelial surfaces. Two weeks after Av1Luc1 treatment of nu/nu mice, immunostaining for SP-A, SP-B, and proSP-C was similar to those patterns observed prior to adenoviral administration. In immunocompetent wild-type mice, however, immunostaining for surfactant proteins was absent in areas associated with chronic lymphocytic infiltration. The recombinant adenoviral vector, Av1Luc1, caused acute inflammatory responses in the respiratory epithelium with disruption of surfactant protein homeostasis in both wild-type and nu/nu mice. Alterations in surfactant homeostasis persisted in wild-type mice. Thus, both acute and thymic-dependent immune responses limit transgene expression and disrupt surfactant protein gene expression and homeostasis. Because surfactant proteins are critical to host defense and to the maintenance of alveolar stability following injury, these findings raise concerns regarding both acute and chronic toxicity of first-generation recombinant adenoviral vectors for gene transfer.

We used immunohistochemical analysis to localize thyroid transcription factor-1 (TTF-1), hepatocyte nuclear factor-3beta (HNF-3beta), prosurfactant proteins B and C (pro-SP-B, pro-SP-C), surfactant protein B (SP-B), and Clara cell secretory protein (CCSP) in developing mouse lung. TTF-1 and HNF-3beta were expressed at the onset of lung morphogenesis (gestational Day 10) and throughout fetal lung development, being detected in the nuclei of airway epithelial cells. TTF-1 was most prominent in distal airway epithelial cells in embryonic lung and HNF-3beta in proximal bronchial and bronchiolar epithelial cells. Pro-SP-B and pro-SP-C were first detected on gestational Day 11, being localized to the cytoplasm of airway epithelial cells. Expression of both pro-proteins was confined to distal airway epithelial cells from gestational Day 12 to Day 16. From gestational Day 17 and thereafter, pro- SP-B was detectable in Type II cells and bronchiolar epithelial cells, whereas pro-SP-C was restricted to Type II cells. SP-B peptide was first detected on gestational Day 17 in the cytoplasm of Type II cells and within the lumen of distal airways. SP-B peptide was detectable only in the cytoplasm of Type II cells in adult lung. CCSP was first detected on gestational Day 17, being localized to the cytoplasm of columnar epithelial cells lining the conducting airways. Pro-SP-B, SF-B, pro-SP-C, and CCSP staining increased before birth. The early expression of TTF-1 and HNF-3beta, preceding and overlapping that of pro-SP-B, mature SP-B, pro-SP-C, and CCSP, supports a regulatory role for TTF-1 and HNF-3beta in lung-specific gene expression.