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The development of selected reaction monitoring methods for targeted proteomics via empirical refinement.
Bereman MS, MacLean B, Tomazela DM, Liebler DC, MacCoss MJ
(2012) Proteomics 12: 1134-41
MeSH Terms: Amino Acid Sequence, Calibration, Humans, Infant, Newborn, Lung, Mass Spectrometry, Molecular Sequence Data, Peptides, Proteomics, Pulmonary Surfactant-Associated Protein B, Reference Standards, Reproducibility of Results, Respiratory Distress Syndrome, Newborn, Sensitivity and Specificity, Software
Show Abstract · Added March 20, 2014
Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation. The ultimate goal is to identify the best performing peptide in terms of ionization efficiency, reproducibility, specificity, and chromatographic characteristics to monitor as a proxy for protein abundance. It is important to emphasize that this method allows the user to perform this refinement procedure in the sample matrix and organism of interest with the instrumentation available. Finally, the method is demonstrated in a case study to determine the best peptide to monitor the abundance of surfactant protein B in lung aspirates.
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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15 MeSH Terms
The Rho pathway mediates transition to an alveolar type I cell phenotype during static stretch of alveolar type II cells.
Foster CD, Varghese LS, Gonzales LW, Margulies SS, Guttentag SH
(2010) Pediatr Res 67: 585-90
MeSH Terms: Alveolar Epithelial Cells, Amides, Biomarkers, Caveolin 1, Cell Differentiation, Cell Shape, Cells, Cultured, Claudins, Gestational Age, Humans, Lung, Mechanotransduction, Cellular, Membrane Proteins, Pepsinogen C, Phenotype, Plasminogen Activator Inhibitor 1, Protein Kinase Inhibitors, Pulmonary Surfactant-Associated Protein B, Pyridines, Stress Fibers, Time Factors, rho-Associated Kinases, rhoA GTP-Binding Protein
Show Abstract · Added January 20, 2015
Stretch is an essential mechanism for lung growth and development. Animal models in which fetal lungs have been chronically over or underdistended demonstrate a disrupted mix of type II and type I cells, with static overdistention typically promoting a type I cell phenotype. The Rho GTPase family, key regulators of cytoskeletal signaling, are known to mediate cellular differentiation in response to stretch in other organs. Using a well-described model of alveolar epithelial cell differentiation and a validated stretch device, we investigated the effects of supraphysiologic stretch on human fetal lung alveolar epithelial cell phenotype. Static stretch applied to epithelial cells suppressed type II cell markers (SP-B and Pepsinogen C, PGC), and induced type I cell markers (Caveolin-1, Claudin 7 and Plasminogen Activator Inhibitor-1, PAI-1) as predicted. Static stretch was also associated with Rho A activation. Furthermore, the Rho kinase inhibitor Y27632 decreased Rho A activation and blunted the stretch-induced changes in alveolar epithelial cell marker expression. Together these data provide further evidence that mechanical stimulation of the cytoskeleton and Rho activation are key upstream events in mechanotransduction-associated alveolar epithelial cell differentiation.
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23 MeSH Terms
Opposing regulation of human alveolar type II cell differentiation by nitric oxide and hyperoxia.
Johnston LC, Gonzales LW, Lightfoot RT, Guttentag SH, Ischiropoulos H
(2010) Pediatr Res 67: 521-5
MeSH Terms: Alveolar Epithelial Cells, Biomarkers, Cell Differentiation, Cell Hypoxia, Cells, Cultured, Cyclic GMP, Cyclic Nucleotide Phosphodiesterases, Type 5, Gene Expression Regulation, Gestational Age, Guanylate Cyclase, Humans, Nitric Oxide, Nuclear Proteins, Oxygen, Pulmonary Surfactant-Associated Protein B, Pulmonary Surfactant-Associated Protein C, RNA, Messenger, Receptors, Cytoplasmic and Nuclear, Signal Transduction, Soluble Guanylyl Cyclase, Thyroid Nuclear Factor 1, Time Factors, Transcription Factors
Show Abstract · Added January 20, 2015
Clinical trials demonstrated decreasing rates of bronchopulmonary dysplasia in preterm infants with hypoxic respiratory failure treated with inhaled nitric oxide (iNO). However, the molecular and biochemical effects of iNO on developing human fetal lungs remain vastly unknown. By using a well-characterized model of human fetal alveolar type II cells, we assessed the effects of iNO and hyperoxia, independently and concurrently, on NO-cGMP signaling pathway and differentiation. Exposure to iNO increased cGMP levels by 40-fold after 3 d and by 8-fold after 5 d despite constant expression of phosphodiesterase-5 (PDE5). The levels of cGMP declined significantly on exposure to iNO and hyperoxia at 3 and 5 d, although expression of soluble guanylyl cyclase (sGC) was sustained. Surfactant proteins B and C (SP-B, SP-C) and thyroid transcription factor (TTF)-1 mRNA levels increased in cells exposed to iNO in normoxia but not on exposure to iNO plus hyperoxia. Collectively, these data indicate an increase in type II cell markers when undifferentiated lung epithelial cells are exposed to iNO in room air. However, hyperoxia overrides these potentially beneficial effects of iNO despite sustained expression of sGC.
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23 MeSH Terms
Posttranslational regulation of surfactant protein B expression.
Guttentag S
(2008) Semin Perinatol 32: 367-70
MeSH Terms: Dimerization, Gene Expression Regulation, Glycosylation, Humans, Peptide Hydrolases, Protein Folding, Protein Precursors, Protein Processing, Post-Translational, Proteolipids, Pulmonary Alveoli, Pulmonary Surfactant-Associated Protein B
Show Abstract · Added January 20, 2015
Although a minor constituent by weight, surfactant protein B (SP-B) plays a major role in surfactant function. It is the unique structure of SP-B that promotes permeabilization, cross-linking, mixing, and fusion of phospholipids, facilitating the proper structure and function of pulmonary surfactant as well as contributing to the formation of lamellar bodies. SP-B production is a complex process within alveolar type 2 cells and is under hormonal and developmental control. Understanding the posttranslational events in the maturation of SP-B may provide new insight into the process of lamellar body formation and into the pathophysiology of pulmonary disorders associated with surfactant abnormalities.
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Developmental and genetic regulation of human surfactant protein B in vivo.
Hamvas A, Heins HB, Guttentag SH, Wegner DJ, Trusgnich MA, Bennet KW, Yang P, Carlson CS, An P, Cole FS
(2009) Neonatology 95: 117-24
MeSH Terms: Adult, Amniotic Fluid, Bronchoalveolar Lavage Fluid, DNA Mutational Analysis, Exudates and Transudates, Female, Gene Expression Regulation, Genetic Predisposition to Disease, Gestational Age, Humans, Infant, Infant, Newborn, Infant, Premature, Male, Polymorphism, Single Nucleotide, Protein Precursors, Proteolipids, Pulmonary Surfactant-Associated Protein B, Respiratory Distress Syndrome, Newborn, Survival Rate, Trachea
Show Abstract · Added January 20, 2015
BACKGROUND - Genetic and developmental disruption of surfactant protein B (SP-B) expression causes neonatal respiratory distress syndrome (RDS).
OBJECTIVES - To assess developmental and genetic regulation of SP-B expression in vivo.
METHODS - To evaluate in vivo developmental regulation of SP-B, we used immunoblotting to compare frequency of detection of mature and pro-SP-B peptides in developmentally distinct cohorts: 24 amniotic fluid samples, unfractionated tracheal aspirates from 101 infants >or=34 weeks' gestation with (75) and without (26) neonatal RDS, and 6 nonsmoking adults. To examine genetic regulation, we used univariate and logistic regression analyses to detect associations between common SP-B (SFTPB) genotypes and SP-B peptides in the neonatal RDS cohort.
RESULTS - We found pro-SP-B peptides in 24/24 amniotic fluid samples and in 100/101 tracheal aspirates from newborn infants but none in bronchoalveolar lavage from normal adults (0/6) (p < 0.001). We detected an association (p = 0.0011) between pro-SP-B peptides (M(r) 40 and 42 kDa) and genotype of a nonsynonymous single nucleotide polymorphism at genomic position 1580 that regulates amino-terminus glycosylation.
CONCLUSIONS - Pro-SP-B peptides are more common in developmentally less mature humans. Association of genotype at genomic position 1580 with pro-SP-B peptides (M(r) 40 and 42 kDa) suggests genetic regulation of amino terminus glycosylation in vivo.
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Pepsinogen C proteolytic processing of surfactant protein B.
Gerson KD, Foster CD, Zhang P, Zhang Z, Rosenblatt MM, Guttentag SH
(2008) J Biol Chem 283: 10330-8
MeSH Terms: Amino Acid Sequence, Bronchi, Humans, Lung, Models, Biological, Molecular Sequence Data, Pepsinogen C, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Pulmonary Surfactant-Associated Protein B, RNA, Small Interfering, Recombinant Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypsin
Show Abstract · Added January 20, 2015
Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to lamellar body genesis in alveolar epithelial type 2 cells. The bioactive, mature SP-B is derived from multistep post-translational proteolysis of a larger proprotein. The identity of the proteases involved in carboxyl-terminal cleavage of proSP-B remains uncertain. This cleavage event distinguishes SP-B production in type 2 cells from less complete processing in bronchiolar Clara cells. We previously identified pepsinogen C as an alveolar type 2 cell-specific protease that was developmentally regulated in the human fetal lung. We report that pepsinogen C cleaved recombinant proSP-B at Met(302) in addition to an amino-terminal cleavage at Ser(197). Using a well described model of type 2 cell differentiation, small interfering RNA knockdown of pepsinogen C inhibited production of mature SP-B, whereas overexpression of pepsinogen C increased SP-B production. Inhibition of SP-B production recapitulated the SP-B-deficient phenotype evident by aberrant lamellar body genesis. Together, these data support a primary role for pepsinogen C in SP-B proteolytic processing in alveolar type 2 cells.
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15 MeSH Terms
Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease.
Griese M, Schumacher S, Tredano M, Steinecker M, Braun A, Guttentag S, Beers MF, Bahuau M
(2005) Respir Res 6: 80
MeSH Terms: Adolescent, Biomarkers, Bronchitis, Chronic, Bronchoalveolar Lavage Fluid, Child, Child, Preschool, Female, Gene Expression Profiling, Humans, Hydrophobic and Hydrophilic Interactions, Infant, Infant, Newborn, Male, Pulmonary Surfactant-Associated Protein B, Pulmonary Surfactant-Associated Protein C, Reproducibility of Results, Sensitivity and Specificity
Show Abstract · Added January 20, 2015
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.
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17 MeSH Terms
Defective surfactant secretion in a mouse model of Hermansky-Pudlak syndrome.
Guttentag SH, Akhtar A, Tao JQ, Atochina E, Rusiniak ME, Swank RT, Bates SR
(2005) Am J Respir Cell Mol Biol 33: 14-21
MeSH Terms: Animals, Blotting, Western, Bronchoalveolar Lavage, Capillaries, Densitometry, Disease Models, Animal, Hermanski-Pudlak Syndrome, Humans, Immunoblotting, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Fluorescence, Phospholipids, Pulmonary Surfactant-Associated Protein A, Pulmonary Surfactant-Associated Protein B, Pulmonary Surfactant-Associated Protein C, Pulmonary Surfactant-Associated Protein D, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, Surface-Active Agents, Time Factors
Show Abstract · Added January 20, 2015
Hermansky-Pudlak syndrome (HPS) in humans represents a family of disorders of lysosome-related organelle biogenesis associated with severe, progressive pulmonary disease. Human case reports and a mouse model of HPS, the pale ear/pearl mouse (ep/pe), exhibit giant lamellar bodies (GLB) in type II alveolar epithelial cells. We examined surfactant proteins and phospholipid from ep/pe mice to elucidate the process of GLB formation. The 2.8-fold enrichment of tissue phospholipids in ep/pe mice resulted from accumulation from birth through adulthood. Tissue surfactant protein (SP)-B and -C were increased in adult ep/pe mice compared with wild-type mice (WT), whereas SP-A and -D were not different. Large aggregate surfactant (LA) from adult ep/pe mice had decreased phospholipid, SP-B, and SP-C, with no differences in SP-A and -D compared with WT. Although LA from ep/pe animals exhibited an increased total protein-to-total phospholipid ratio compared with WT, surface tension was not compromised. Phospholipid secretion from isolated type II cells showed that basal and stimulated secretion from ep/pe cells were approximately 50% of WT cells. Together, our data indicate that GLB formation is not associated with abnormal trafficking or recycling of surfactant material. Instead, impaired secretion is an important component of GLB formation in ep/pe mice.
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23 MeSH Terms
Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung.
Brasch F, Ochs M, Kahne T, Guttentag S, Schauer-Vukasinovic V, Derrick M, Johnen G, Kapp N, Muller KM, Richter J, Giller T, Hawgood S, Buhling F
(2003) J Biol Chem 278: 49006-14
MeSH Terms: Amino Acid Sequence, Aspartic Acid Endopeptidases, Humans, Immunohistochemistry, Lung, Microscopy, Immunoelectron, Molecular Sequence Data, Protein Processing, Post-Translational, Pulmonary Surfactant-Associated Protein B, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added January 20, 2015
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.
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10 MeSH Terms
Surfactant protein B in type II pneumocytes and intra-alveolar surfactant forms of human lungs.
Brasch F, Johnen G, Winn-Brasch A, Guttentag SH, Schmiedl A, Kapp N, Suzuki Y, Müller KM, Richter J, Hawgood S, Ochs M
(2004) Am J Respir Cell Mol Biol 30: 449-58
MeSH Terms: Adult, Animals, Bronchoalveolar Lavage Fluid, Cells, Cultured, Golgi Apparatus, Humans, In Vitro Techniques, Lung, Male, Microscopy, Immunoelectron, Myelin Sheath, Protein Precursors, Protein Processing, Post-Translational, Proteolipids, Pulmonary Alveoli, Pulmonary Surfactant-Associated Protein A, Pulmonary Surfactant-Associated Protein B, Pulmonary Surfactants, Rats, Rats, Wistar, Subcellular Fractions
Show Abstract · Added January 20, 2015
Surfactant protein B (SP-B) is synthesized by type II pneumocytes as a proprotein (proSP-B) that is proteolytically processed to an 8-kD protein. In human type II pneumocytes, we identified not only proSP-B, processing intermediates of proSP-B, and mature SP-B, but also fragments of the N-terminal propeptide. By means of immunoelectron microscopy, proSP-B and processing intermediates were localized in the endoplasmic reticulum, Golgi vesicles, and few multivesicular bodies in type II pneumocytes in human lungs. A colocalization of fragments of the N-terminal propeptide and mature SP-B was found in multivesicular, composite, and some lamellar bodies. Mature SP-B was localized over the projection core of lamellar bodies and core-like structures in tubular myelin figures. In line with immunoelectron microscopy and Western blot analysis of human type II pneumocytes, a fragment of the N-terminal propeptide was also detected in isolated rat lamellar bodies. In conclusion, our data indicate that the processing of proSP-B occurs between the Golgi complex and multivesicular bodies and provide evidence that a fragment of the N-terminal propeptide and mature SP-B are transported together to the lamellar bodies. In human lungs, mature SP-B is involved in the structural organization of lamellar bodies and tubular myelin by the formation of core particles.
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21 MeSH Terms