Biomedical subjects

Susan Guttentag

Publications and source records attributed to Susan Guttentag.

8 recordsLinked to original sources

PubMed2005

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease.

BACKGROUND: Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects. METHODS: We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19). RESULTS: Pro-SP-B of 25-26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19-21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening. CONCLUSION: Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.

Adolescent↗

PubMed2005

Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung.

Branching morphogenesis in the lung serves as a model for the complex patterning that is reiterated in multiple organs throughout development. Beta-catenin and Wnt signaling mediate critical functions in cell fate specification and differentiation, but specific functions during branching morphogenesis have remained unclear. Here, we show that Wnt/beta-catenin signaling regulates proximal-distal differentiation of airway epithelium. Inhibition of Wnt/beta-catenin signaling, either by expression of Dkk1 or by tissue-specific deletion of beta-catenin, results in disruption of distal airway development and expansion of proximal airways. Wnt/beta-catenin functions upstream of BMP4, FGF signaling, and N-myc. Moreover, we show that beta-catenin and LEF/TCF activate the promoters of BMP4 and N-myc. Thus, Wnt/beta-catenin signaling is a critical upstream regulator of proximal-distal patterning in the lung, in part, through regulation of N-myc, BMP4, and FGF signaling.

Animals↗

PubMed2004

Developmental regulation of claudin localization by fetal alveolar epithelial cells.

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Pepsinogen C: a type 2 cell-specific protease.

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.

Biomarkers↗

PubMed2003

Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung.

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.

Amino Acid Sequence↗

PubMed2003

Surfactant protein A and B genetic variants in respiratory distress syndrome in singletons and twins.

Interactive genetic and environmental factors may influence the differentiation of surfactant and the risk of respiratory distress syndrome (RDS). DNA samples from 441 premature singleton infants and 480 twin or multiple infants were genotyped for surfactant-specific protein (SP)-A1, SP-A2, and SP-B exon 4 polymorphisms and intron 4 size variants in a homogeneous white population. Distributions of the SP-A and SP-B gene variants between RDS and no-RDS infants were determined alone and in combination. SP-A1 allele 6A2 (p = 0.009) and the homozygous genotype 6A2/6A2 (p = 0.003) were overrepresented in RDS of singletons when the SP-B exon 4 genotype was Thr/Thr, and underrepresented in RDS of multiples when the SP-B genotype was Ile/Thr (p = 0.012 for 6A2 and p = 0.03 for 6A2/6A2) or Thr/Thr (p = 0.12 for 6A2 and p = 0.018 for 6A2/6A2, respectively). The SP-A 6A2 allele in the SP-B Thr131 background predisposed the smallest singleton infants to RDS, whereas near-term multiples were protected from RDS. There was a continuous association between fetal mass and risk of RDS, defined by the SP-A and SP-B variants. Labeled lung explants with the Thr/Thr genotype showed proSP-B amino-terminal glycosylation, which was absent in Ile/Ile samples. Genetic and environmental variation may influence intracellular processing of surfactant complex and the susceptibility to RDS.

Diseases in Twins↗

PubMed2003

Cysteine protease activity is required for surfactant protein B processing and lamellar body genesis.

Surfactant protein (SP)-B is essential for lamellar body genesis and for the final steps in proSP-C post-translational processing. The mature SP-B protein is derived from multistep processing of the primary translation product proSP-B; however, the enzymes required for these events are currently unknown. Recent ultrastructural colocalization studies have suggested that the cysteine protease Cathepsin H may be involved in proSP-B processing. Using models of isolated human type 2 cells in culture, we describe the effects of cysteine protease inhibition by E-64 on SP-B processing and type 2 cell differentiation. Pulse-chase labeling and Western immunoblotting studies showed that the final step of SP-B processing, specifically cleavage of SP-B(9) to SP-B(8), was significantly inhibited by E-64, resulting in delayed accumulation of SP-B(8) without adverse effects on SP-A or glyceraldehyde phosphate dehydrogenase expression. E-64 treatment during type 2 cell differentiation mimicked features of inherited SP-B deficiency in humans and mice, specifically disrupted lamellar body genesis, and aberrant processing of proSP-C. Reverse transcriptase-polymerase chain reaction and Western immunoblotting studies showed that Cathepsin H is induced during in vitro differentiation of type 2 cells and localizes with SP-B in multivesicular bodies, composite bodies, and lamellar bodies by immunoelectron microscopy. Furthermore, Cathepsin H activity was specifically inhibited in a dose-dependent fashion by E-64. Our data show that a cysteine protease is involved in SP-B processing, lamellar body genesis, and SP-C processing, and suggest that Cathepsin H is the most likely candidate protease.

Base Sequence↗

PubMed2002

Notch1 and Jagged1 expression by the developing pulmonary vasculature.

The molecular mechanisms of pulmonary vascular development are poorly understood. Cell-specific developmental pathways are influenced by cell-cell signaling. Notch signaling molecules are highly conserved receptors active in many cell-fate determination systems. Recent observations of Notch molecules and a Notch ligand, Jagged1, suggest their importance in vascular morphogenesis, and particularly pulmonary vascular development. We performed a systematic evaluation of Notch1/Jagged1 gene and protein expression in the developing mouse lung from embryonic day 11 until adulthood by using quantitative PCR, immunofluorescence, and electron microscopic analysis. mRNA transcripts for Notch1-4 and Jagged1 increased progressively from early to later lung development, accompanied by a simultaneous rise in endothelial cell-specific gene expression, a pattern not seen in other organs. Notch1 mRNA was identified on both epithelial and mesenchymal structures of the embryonic lung. Immunofluorescence staining revealed the progressive acquisition of Notch1 and Jagged1 proteins by the emerging endothelium. Notch1 and Jagged1 were seen initially on well-formed, larger vessels within the embryonic lung bud and progressively on finer vascular networks. Each was also expressed on surrounding nonvascular structures. The localization of Notch1 and Jagged1 on endothelial cell surface membranes within the alveolar microvasculature was confirmed by immuno-electron microscopy. These temporal and spatial patterns in Notch1/Jagged1 gene and protein expression suggest multiple potential paths of cell-cell signaling during lung development and vascular morphogenesis.

Animals↗